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THE INFLUENCE OF HEATED SOILS ON SEED GER- 
MINATION AND SOIL GROWTH 



JAMES JOHNSON 

UNIVERSITY- C^ 
] HESiS {Oii± 



Department of HortictiUiire, W'/.STons/?i Agricultural Experirnnit Station 



Reprinted from 

Soil Science, Vol. VII, No. 1, January, 1919 



n. of »>• 

JAN 10 1920 



.of 



Reprinted from Soil Science, 
Vol. VII, No. 1, January, 1919 



THE INFLUENCE OF HEATED SOILS ON SEED GERMINATION 
AND PLANT GROWTH 

JAMES JOHNSON 

Department of Horticulture, Wisconsin Agricultural Experiment Station 

Received for publication January 6, 1918 

INTRODUCTION 

The use of heat-sterilized soils in methods of research in soil biology and 
plant pathology as well as in various phases of practical agriculture is rapidly 
increasing in value. For purposes of research especially, the sterilization of 
soil by heat is practically the only method which can be relied upon for pur- 
poses of rendering the soil sterile as regards any particular organism. Since 
the application of heat to the soil may, and usually does, result in important 
changes aside from sterilization which may greatly influence plant growth, it 
is evident that a knowledge of the alteration's which occur or may be expected 
to occur in the soil is essential in order to draw reliable conclusions from cer- 
tain types of research conducted with heated soils. The problem is by no 
means a new one. A great variety of literature exists upon the subject of soil 
sterilization from various angles of attack. Since the subject may be treated 
from the standpoint of the soil itself as chemistry, biology, or physics, or from 
the standpoint of plants grown in these soils as physiology or pathology, the 
field has been a very productive one. At the same time, its complexity has 
led to quite widely varying results and conclusions. Although it is evident 
that investigations in this field should take into account all factors which may 
be involved before drawing far-reaching conclusions, it is also evident that it 
is exceedingly difficult to grasp the significance of, and lay proportionate 
weight upon all the factors concerned. The results presented in this paper 
were first undertaken from a phytopathological point of view. The bearing 
of the conclusions drawn on phytopathological research are contemplated in 
another paper. It is the purpose of this paper to present the data obtained 
on the changes produced in heated soils as measured by their influence on 
seed germination and plant growth, and to discuss the probable nature of 
these changes. 

A clear conception of the subject of soil sterilization requires at the outset 
a good understanding of the methods by which soils may be sterilized. These 
are: (a) Sterilization by heat, and (b) sterilization by chemicals. The for- 
mer may again be divided into methods using (a) live steam, and (b) dry 
heat; the latter into methods using (a) volatile antiseptics, and (b) non- 
volatile antiseptics. Clearly these methods may be expected to yield funda- 

1 

SOIL SCIENCE, VOL. VII, NO. 1 



2 JAMES JOHNSON 

mentally different results, not only in regard to the method employed, but 
also in regard to the intensity with which it is applied. Technically the term 
sterilization means the complete destruction of all living matter in a medium, 
and implies the continued sterility of this medium if desired. This term as 
used in connection with the treatment of the soil, however, does not 
necessarily mean the complete destruction of all living matter in the soil nor 
its continued sterihty. Ordinarily it refers to the destruction of one or 
more specific organisms which are not desired in the soil with no special pre- 
cautions to prevent reinfestation. In a broader sense, however, the object 
of soil sterilization is the production of a more favorable medium for the 
growth of cultivated plants by means of heat or chemical disinfection. This 
may be brought about as much by the improvement of the chemical, physical, 
or biological relationships in the soil, as by the simple destruction of one or 
more forms of undesirable organisms. In view of these facts the terms 
"partial sterilization" and "pasteurization" have come to be used by some 
investigators. 

The object of soil sterilization in practical agriculture is, therefore, usually 
either the destruction of some particular plant parasite harbored in the soil, 
or the improvement of the fertihty of the soil by affecting its chemical, 
biological or physical condition. Where it is practiced for the former reason, 
the latter results are naturally also secured as secondary effects. Many 
farmers are, for instance, coming to regard the process as valuable from the 
standpoint of destruction of weed seeds in plant beds alone. 

Various methods of appHcation have been devised with the result that the 
practice has been increasing rapidly in certain forms of intensive plant culture. 
Up to the present time, soil sterilization has been found most useful and has 
received its greatest impetus in the culture of plants under glass. The vegetable 
forcing house industry particularly, has used the method on a large scale. 
Florists use it less commonly, but find it advantageous for certain plants and 
for starting seed. In the culture of tobacco, ginseng, coniferous seedlings, 
and most seedlings for the market gardener, it has been found well worthy of 
use. 

BRIEF SUMMARY OF LITERATURE 

The beneficial action of heated soils upon plant growth was observed long 
before the sterilizing value of heat was known. According to Sir Humphrey 
Davy (12) the improvement of "sterile" lands by burning was known to the 
ancient Romans, the custom having been mentioned by Virgil in his first book 
of the Georgics. It is well known that the burning of various types of soil 
became quite general in Europe during the eighteenth century. This method 
was accompanied by a considerable amount of scientific investigation upon the 
subject, until the practice fell into disuse about the middle of the last century. 

The actual use of sterilizing agents upon the soil for the primary purpose of 
destroying injurious forms of hving organisms present in the soil is largely a 



INFLUENCE OF HEATED SOILS ON GERMINATION .\ND GROWTH 6 

development of the past three or four decades. Accompanying these studies 
and practices of soil sterilization for the destruction of soil organisms, there 
has accumulated a great deal of literature dealing with the scientific principles 
involved. These publications have been especially stimulated by a desire to 
explain the reason for the increased growth of plants on sterilized soils. The 
investigations undertaken from quite different angles of attack and with great 
variation in type of soil, kind and intensity of sterilizing agents, as well as in 
type of plants used, have quite naturally resulted in widely varying con- 
clusions. 

A detailed review of the literature concerning soil sterihzation would be too 
voluminous to present here. For present purposes it may suffice to present 
in brief form an outline of the principal changes produced in and by steri- 
lized soils, and to mention briefly certain theories concerning the nature of 
the action of sterilized soils on plant growth, leaving the discussion of the 
literature more directly concerned with this investigation to be treated under 
the separate phases of the subject as they are taken up. 

Principal changes produced in and by sterilized soils. 

I. Destruction of life. 

A. Normal soil flora and fauna, desirable and undesirable forms of bacteria, fungi, pro- 

tozoa, and higher animals. 

B. Plant parasites, especially pathogenic bacteria, fungi, nematodes, and injurious 

soil infesting insects. 

C. Propagalive organs of higher plants, especially weed seeds. 

II. Immediate chemical action (formation of toxic and beneficial compounds). 

A. Decomposition of organic material resulting in formation of ammonia, carbon diox- 

ide and various new and complek organic compounds. 

B. Decomposition of inorganic material, reduction of nitrates and nitrites to ammonia 

and increased solubility of potassium, phosphorus and other salts. 

III. Bio-chemical action. 

A. Increased ammonification particularly and modified nitrification, denitrification, 
and nitrogen fixation. 

IV. Physical action. 

A. Absorptive capacity of soil modified for water, gases, and salts. 

B. Increased concentration of soil soltdion. 

C. Modified capillarity, colloidal state, and mechanical condition. 

D. Modified color and odor. 

V. Action on organisms growing in sterilized soils. 

A. Lower organisms. 

1. Increased development due to reduced competition, increased food supply, 

destruction of "bacterio- toxins," "stimulation" by products added or 
formed, or other causes. 

2. Retardation in growth in rare cases due to injurious conditions produced. 

B. Green plants. 

1 . Injurious action as indicated by retarded rate and percentage of seed germi- 

nation and by retarded rate of plant growth. 

2. Beneficial action as shown by increased rate and percentage of seed germina- 

tion and increased rate and amount of plant growth. 

3. Modification in form, color, and other "qualitative" changes. 



4 JAMES JOHNSON 

The theories which have been promulgated by various investigators rest 
primarily upon the explanation of the beneficial and injurious action of ster- 
ilized soils upon plant growth. These theories are partially interrelated and 
do not permit of satisfactory classification, but the main ones may be grouped 
as follows: 

1. Stimulation hypothesis. Koch's (31) theory formulated in 1899 was 
probably the first attempt at scientific explanation of observed facts. He 
believed the sterilizing agent or its products directly ''stimulated" the growth 
of the plants or soil bacteria, which in turn influenced plant growth. 

2. Modified bacterial activity. Hiltner and Stormer (25) in 1903 showed by 
soil bacterial counts that sterilization resulted in an initial decrease in numbers 
of organisms followed by a marked increase in numbers, and hence an increase 
in the productiveness of the soil. 

3. Protozoan theory. Russell and Hutchinson (62) believe that the benefit 
from "partial sterilization" may result from the destruction of the larger 
phagocytic microorganisms (mostly protozoans) which inhibit the develop- 
ment of the beneficial organisms. The protozoa being destroyed, the am- 
monifying bacteria, for instance, increase rapidly producing an increase in 
nitrogen and hence plant growth. 

4. "Bacierio-toxin" theory. Grieg-Smith (22) considers that certain sub- 
stances which he calls "bacterio-toxins" and which exist naturally in soils, 
inhibiting bacterial growth, are destroyed by sterilizing agents. 

5. Modified organic soil compounds. Schreiner and Lathrop (65) believed 
that certain complex organic soil constituents produced as a result of sterili- 
zation by heat, may increase plant growth while others inhibit it. That or- 
ganic soil constituents of an indefinite nature were produced which were in- 
jurious to plant growth in heated soils had also been suggested by Struckman 
(73), Dietrich (13) and Pickering (52). 

6. Modified inorganic soil compounds. This theory though supported by 
no investigators in particular should be added here since it has been repeat- 
edly shown since the time of Struckman (73) that increase in inorganic plant 
food constituents occurs in heated soils. The theory is perhaps best supported 
by Liebscher (40) who believed that sterilization was essentially nitrogenous 
fertilization. 

7. Plant parasite theories. The fact that in certain soils the benefit of soil 
sterilization may be due largely to the destruction of parasitic organisms is un- 
questionable. The wide appHcation of this theory, however, to the subject 
in question, as supported perhaps most energetically by Bolley (5), serves to 
place this type of response to sterilization among the theories explaining the 
action of sterilized soils. 

8. Physical theories. These theories are not subscribed to by any author 
in particular at the present time, although it was quite generally believed at 
one time that all the benefit derived from burning the soil was due to purely 
physical changes. Some of the physical factors which play a part in soil fer- 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 5 

tility are, however, coming to be regarded as very influential in conjunction 
with chemical factors. Seaver and Clark's (67) "concentration theory" may, 
for instance, properly be placed here. 

For a more detailed summary of the subject of soil sterilization especially 
as regards the relation of protozoa to sterilized soils, the recent review of 
Kopelofif and Coleman (34) should be consulted. 

EXPERIMENTAL WORK 

The investigations upon the subject of soil sterilization were begun by 
the writer in 1909 with the object of studying methods for the control of 
the damping-oflf disease of plants, caused by Pythium debaryanum, and 
Rhizoctonia. At the outset, it became evident that when soils were treated 
either with heat or chemicals, various "secondary effects" of sterilization oc- 
curred, which modified the soil and plant growth aside from the control of dis- 
ease. Following a publication upon the control of damping-off in plant beds 
(27), the study of these "secondary effects" was undertaken. The primary 
object of this study was to attempt to find an explanation for the injurious 
action followed by the beneficial action of steriHzed soils on plant growth. On 
account of the size and complexity of the problem, it became necessary to 
limit the investigation primarily to the effects of sterilization by heat. The 
studies up to date have been mainly concerned with the action of heated soils 
upon seed germination and plant growth; although various other phases have 
been taken up from time to time with the general idea of rounding out the 
problem in such a way as to make it of value also in the plant pathological 
investigations carried on simultaneously. In carrying on this work the soil has 
in many cases been heated to a degree far above that which is used in ordinary 
sterilization. While no apology may be necessary for such procedure from a 
chemical standpoint, the general idea has been to increase the action so that 
there would be produced a sufficient magnification of effect to obtain a good 
theoretical working basis for the explanation of similar results secured at or- 
dinary temperatures of sterilization. While comparisons cannot readily be 
made with the "partial sterilization" of Russell and his associates, (62, 64), 
yet it is hoped that some evidence of value in this connection may be gathered 
from the results. The work presented here is perhaps more comparable to 
that of Pickering (50-53), or Seaver and Clark (67, 68), than to that of other 
investigators. On the other hand the writer has gone into the subject with a 
desire to bring together the various phases of the subject as presented in the 
literature with the idea of rounding out the problem and attempting to clear 
up some of the obscure points. 

Materials and methods 

The different soils used were selected largely for their variations in general 
type, especially in physical structure, rather than for differences in their chemi- 



JAMES JOHNSON 



cal properties or their productivity. Their names will, therefore, quite satis- 
factorily indicate the general nature of these soils. No complete mechanical 
analyses have been made, but a determination of the loss on ignition indicates 
the range of organic matter present in the various soils as well as the general 
character of each soil in regard to this constituent. The determinations of 
total nitrogen, phosphorus, and potassium serve to give an idea of the state of 
fertility of these soils as far as these elements are concerned. The general 
character of the soils used is summarized in table 1.^ 

TABLE 1 

General character of sails used in the experimental work 





LOSS ON 












NAME 


IGNI- 
TION 


N 


P2O5 


K2O 


SOURCE 


GENERAL DESCRIPTION 




per cent 


percent 


percent 


Percent 






Waukesha silt loam 


5.4 


0.26 


0.056 


1.65 


Milton, Wis- 
consin 


Dark, tillable, fairly 
productive 


Muck 


14.8 


0.65 


0.14 


1.52 


Station farm, 
Madison 


From drained cropped 




land, fairly fertile 














but responding to 














nitrogen 


Virgin sandy loam 


5.6 


0.26 


0.054 


0.66 


Station farm, 
Madison 


Dark, good texture, 
not fertile from 
wooded pasture land 


Miami silt loam 




0.20 


0.07 


1.69 


Station farm, 
Madison 


From field cropped 10 
yekrs to tobacco but 
heavily fertilized 


Fine sandy loam 


4.3 


0.14 


0.068 


1.20 


Station or- 
chard, Mad- 
ison 


A medium light soil 
containing consider- 
able silt, not very 
productive 


Peat 


68.1 


2.82 






Station farm, 
Madison 


A thoroughly decom- 




posed peat, previ- 














ously cropped and 














quite fertile 


Norfolk sand 


1.9 


0.05 






Upper Marl- 


A very light sand, rel- 












boro, Mary- 


atively low in pro- 












land 


ductivity 


Red clay 


5.5 


02 






Ashland, Wis- 


Very heavy red clay 










consin 


and non-productive 



The soils were taken from the fields during the fall and allowed to air dry for 
most of the work where dry heat was used. The methods of heating the soils 
were usually of two kinds : First by steam in the autoclave at which a temper- 
ature of 114°-116°C. was maintained for one and one-half hour, or second by 
dry heat, in gas, electric, or hot water ovens. For temperatures of 500°C. or 

1 The writer is indebted to the Soils Department, University of Wisconsin, for the deter- 
minations of nitrogen, potassium, and phosphorus. 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 7 

over the electric furnace was used. The time of heating was usually brought 
as near to two hours as possible. 

When the work was first started and in fact during the greater part of this 
investigation, it was supposed that the time of heating the soil was of com- 
paratively small consequence. The soil was usually, therefore, kept at the 
desired temperature for only a comparatively short period of time, the heat 
being allowed to increase gradually in the dry ovens for the first hour. Later 
on it became evident that a source of experimental error lay in the length of 
time of heating and the lack of uniformity of heat distribution in large 
samples of soil. Where large quantities of soil were heated, they were usually 
spread out in the ovens in a layer about one inch thick. This was also true 
when heating was done in the autoclave for short periods of time. The 
temperatures were taken either by means of a mercury or electrical ther- 
mometer for low temperatures, or with a Pyrometer for temperatures above 
250°C. In the later experiments, a mercury thermometer was also used 
for temperatures up to 500°C. It was deemed important to know ap- 
proximately the rate of rise of the temperature of the soils in the autoclave. 
An arrangement was made by means of a packing valve and electrical re- 
sistance thermometers by which this could be done. The rise in temperature 
of substances placed in the autoclave was relatively rapid at first but reached 
the maximum slowly. According to these preliminary tests, 35 to 60 minutes, 
depending on the container used, should be sufficient to thoroughly heat the 
soil throughout in the autoclave to a temperature of 115°C. at 15 to 20 pounds 
of pressure in bulks as large as 2 to 3 kgm. One hour and thirty minutes was, 
however, always allowed for sterilization in the autoclave except where 
otherwise stated. 

In the germination tests the soils were heated in the Petri dishes used in the 
tests at the lower temperatures, but in clay dishes at the higher temperatures. 
Fifty grams of air-dry soil was weighed out and used in all cases in the germi- 
nation tests. As soon as cool after heating, water was added to the soils bring- 
ing them all as near as possible up to the same percentage of moisture, which 
was practically but not quite up to saturation. As soon as all the soils were 
evenly moistened, 100 seeds, which had previously been counted out, were 
placed in each Petri dish, merely being scattered over the surface of the soil. 
The tests were all run in duplicate. The Petri dishes retained a fairly con- 
stant supply of moisture from 10 to 12 days, and it was rarely found necessary 
to add more water to complete the tests. The seeds were picked out with 
tweezers and the number that germinated at certain intervals, usually of 12, 
18, or 24 hours, were recorded. Checks on unheated soil or filter paper were 
always used. 

In the case of heating large quantities of soil with dry heat for plant growth 
studies, more difficulty was encountered in getting uniformity of heating. In 
some of the earlier tests the soil was heated directly in the pots usedj but later 
it was found more satisfactory to heat the soil in shallow pans which would 



8 JAMES JOHNSON 

permit of one or two stirrings during the heating process as well as a 
thorough mixing afterward. After being thoroughly cooled, the soils were 
watered and sown to seed or seedlings transplanted on the same day or the 
day following heating. No special attempt was made in the ordinary tests 
to prevent reinfestation of the pots with organisms. 

Extraction of the soil with water was made by allowing equal weights of 
water and soil to remain in contact for 24 hours with frequent shaking or stir- 
ring. The extract was then filtered off through filter paper, usually with re- 
duced pressure when filtering was slow. These extracts were used for germi- 
nation tests by saturating three or four layers of filter paper in Petri dishes 
with the solution, using water on filter paper as checks. Extracts obtained 
in this way were also used for the freezing-point determinations and in the 
temperature studies with Dewar flasks. 

Freezing-point determinations were made with a Beckmann thermometer 
in the ordinary way, the method differing, however, from that described by 
Boyoucos (6), in that extracts were used instead of the soil itself. The read- 
ings are not intended to represent the actual concentration of the soil solution 
in the soil as they are no doubt too low, but they do give a fairly good measure 
of the comparative concentrations of the soil solutions used. 

Ammonia determinations were made by the ordinary magnesium oxide dis- 
tillation method, which again is to be regarded as giving comparative rather 
than actual amounts of ammonia present. 

Seed germination on heated soils 

The earlier workers on heated soils did not apparently note any particular 
effect of heated soil on seed germination. This is to be expected since it is 
usually only in comparative germination tests that this fact becomes most 
evident. Stone and Smith (71) were probably the first to carry on experi- 
ments along this line. They found an apparent acceleration of germination 
in heated soils and noted further that different kinds of seeds behaved differ- 
ently in this respect. Pickering (50) started a fairly extensive study of the sub- 
ject of the toxic action of heated soils as measured by seed germination. Un- 
fortunately his first papers, especially, are marked by considerable lack of 
uniformity in results and although most of the general conclusions drawn are 
quite correct in principle, the data are in many cases meager and confusing. 
He found, for instance, in one case that heating soil to 100°C. prevented seed 
germination altogether, while heating to 250°C. had little effect. In reality 
the opposite is more likely to be the case. On the whole, however, he con- 
cludes that the time of incubation increases considerably as the temperature 
to which the soil is heated is increased, whereas the percentage of seeds ger- 
minating decreases. In some cases acceleration was noted on soils heated 
to 60° or 80°C. Heating to 200° produced the maximum retarding effect. 
Seeds varied in their response, mustard for instance being more affected by 
heating than rye. The length of time of heating soil at a certain temperature 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 9 

had no special eflFect, but increased moisture content on heating increased the 
toxicity. Pickering also noted that the toxic property was retained in stor- 
age of heated soils, and that its retention was influenced by moisture content 
and temperature during storage. 

In a second paper Pickering (51) reports that the formation of the inhibitory 
substance to seed germination begins at temperatures as low as 30° C. and 
reaches its maximum at 250°. He found most soils in their natural state less 
favorable for germination than water alone, and that sterilization by antisep- 
tics is equivalent to heating to 60° to 75°C. as far as seed germination is con- 
cerned. The "poverty" or "richness" of a soil did not seem to bear any re- 
lation to its behavior when heated. Takoma soil (obtained from Whitney, 
U. S. Dept. Agr.) was found to be very toxic when heated and to turn seeds 
sown upon it black in color. Pickering attempted to show in these and later 
papers that the toxic property of heated soils was connected with increase in 
soluble organic matter, but without good success, as the exceptions found were 
too numerous. His results on the loss of toxicity in heated soils are especially 
interesting and lead him to conclude that the disappearance of the toxin is due 
to an oxidation process. Pickering's conclusions on the nature of the toxic 
substance will be discussed more in detail in a later paragraph. 

Russell and his collaborators (62-64) used seed germination tests to a con- 
siderable extent as a measure of the changes induced in soils by heating and by 
treatment with volatile antiseptics. These results are hardly to be compared 
with that on soils heated to high temperatures such as those used by Picker- 
ing (50) and in the investigations reported in this paper. Russell and Pether- 
bridge (64) do not agree, however, with Pickering in the supposition that the 
toxic agent necessarily disappears in storage or that it is organic in nature. 

In making seed germination studies on heated soils, it at once becomes evi- 
dent that a number of primary factors are concerned, together with a consider- 
able number of secondary factors, which may influence the results secured in 
such a way as to obscure or exaggerate their importance. The primary factors 
are especially those of temperature to which the soil is heated, type of soil used, 
and kind of seed used in the germination test. These factors are all important 
and fundamental in comparing results. As secondary factors which may be 
considered as influencing or explaining the results secured, the following have 
especially been considered: length of time of heating, length of time between 
heating and the germination test, manner of storage following heating, percent- 
age of moisture at time of heating, method of heating (dry or moist heat) , aeration 
during and after heating, size of soil particles, natural toxicity of the soil, tem- 
perature of germination, aeration during germination test, percentage moisture 
in soil during germination, action of light during germination, size and viabiUty 
of seeds, and time required for normal germination. Although all these fac- 
tors could not be studied in detail, they have been given sufficient consider- 
ation to indicate the part which they might play in the results secured if 
they occurred as unavoidable variable factors. 



10 JAMES JOHNSON 

Temperature of heating. The temperature to which a given soil is heated 
has been found to be "probably the most important factor in seed germination 
tests on heated soils. This is well illustrated in table 2 which shows the rela- 
tive rate of germination of lettuce seed in Waukesha silt loam soil heated to 
temperatures ranging from 50° to 350°C. It may be observed that the toxic 
property of the heated soil begins at 100°C., increases rapidly up to 250°C., 
and finally decreases at higher temperatures, having practically been lost 
on soil heated to 350°C. Minor variations sometimes occur which are 
not shown in this table, i.e., the increased germination at the lower 
temperatures of heating, (table 6), which condition occurs in certain soils be- 
fore the temperature of retardation occurs. This is not to be taken, however, 
as the result of a different type of product forming in the soils heated to the 
lower temperatures from that produced at a somewhat higher temperature 
where retardation begins. It is rather to be considered as the stimulating action 
of a small quantity of the toxic substance produced. It may be, of course, 
assumed here that the low temperatures destroy the naturally present toxic 
property of some soils, but reasons for not assuming this to be so will appear 
later, although most soils do possess a natural element slightly toxic to seed 
germination. It will also be noted in table 2 that the toxic action is exhibited 
in this case quite largely by the retarding influence on germination, the final 
or total germination at the maximum temperature of retardation being as 
great as that on the not-heated soil. This is not, however, always found to 
be the case. More frequently, in fact, the total germination at the maximum 
of retardation is considerably lower than that of the unheated soils, indicat- 
ing that some of the seeds have not been able to recover from the toxic agent 
and are in many instances actually killed. 

A very considerable number of the tables presented here, as well as a num- 
ber which are not included, repeatedly show the same general relation of tem- 
peratures of heating the soil and the efifect on seed germination as stated above. 
The variation for different soils and different seeds is very great as will be 
shown by the following considerations. Although the temperature of 250°C. 
has not been determined with any fine detail as the exact turning point for 
all soils, its almost invariable occurrence on heating to this temperature as 
nearly as possible in gas and electric ovens points quite conclusively to the 
critical temperature for seed germination as lying close to 250°C. 

Soil type. The amount of the toxic action produced on heating is very 
largly dependent upon the soil type used. To illustrate this point, the results 
shown in table 3 are presented. Seven different soils were used, all heated to 
250°C. and their action on germination of lettuce seed compared with the ac- 
tion on unheated checks. A retardation of the germination occurred with 
all the heated soils used as compared with the checks. The extent of this re- 
tardation and the rate of recovery from it is, however, quite different, being 
greatest and slowest on the Waukesha silt loam and least and most rapid on 
the fine sandy loam. Although heating to a different temperature, or 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



11 



using another kind of seed, might have led to results more complicated and 
more difficult to explain, it is worth while noting here that the extent of the 
toxic action does not seem to be correlated with any one common characteris- 

TABLE 2 

Relative rate of germination on soil heated to different temperatures; Waukesha silt loam; 

lettuce seed 



TREATMENT 


GERMINATION AFTER 


TOTAl 




24 hours 


42 hours 


66 hours 


90 hours 


114 hours 


138 hours 




Not heated 


Per cent 
57 
73 

2 








41 


per cent 
89 
91 
50 
25 

6 


77 
92 


per cent 
92 
94 
80 
71 
32 
3 
90 
95 


per cent 

84 
80 
49 
15 


per cent 

87 
82 
57 
27 


Per cent 
90 

85 
67 
35 


per cent 
92 


Heated to 50°C 

Heated to 100°C 

Heated to 150°C 

Heated to 200°C 

Heated to 250°C 

Heated to 300 °C 

H^tedto350°C 


95 
95 
89 
82 
92 
95 
95 



TABLE 3 
Relative rate of germination of seed on different soils heated to 250°C.; lettuce seed 







GERMINATION AFTER 








24 
hours 


42 
hours 


66 

hours 


90 

hours 


114 
hours 


138 
hours 








per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


Peat I 


Check 


68 


85 


87 


89 






89 


Heated 








1 


5 


16 


62 


84 


Muck i 


Check 


66 


83 


85 


87 






87 


Heated 











12 


69 


83 


90 


Waukesha silt loam ( 


Check 
Heated 


73 



88 
2 


89 
3 


90 

7 


IS 


30 


91 
67 


Virgin sandy loam ( 


Check 
Heated 


78 



94 



95 

1 


36 


62 


72 


95 
85 


Fine sandy loam < 


Check 
Heated 


75 



87 
1 


88 

7 


80 


95 


96 


88 
98 


Clay 1 


Check 


70 


81 


83 


86 


87 




87 


Heated 





4 


7 


45 


60 


66 


84 


Norfolk sand s 


Check 
Heated 


77 



87 
4 


88 
8 


46 


69 


82 


88 




92 



tic of the soUs used. Statements have been made, and also contradicted, in 
the Hterature that the amount of the toxic action is in proportion to the 
amount of vegetable matter or humus present in the soil, or that it is corre- 



12 



JAMES JOHNSON 



lated with soil fertility. That the amount of organic matter in a soil plays 
a large part, and that the toxic property is formed from organic matter in one 
form or another is seemingly quite evident. In an attempt to illustrate this, 
the results shown in table 4 -were secured where different amounts of quartz 

TABLE 4 
Influence of content of vegetable matter on the injurious action to seed germination on heating 

to 250°C.: cabbage seed 



PERCENTAGE VEGETABLE MATTER 



Ground quartz 

Quartz with 10 per cent 
peat 

Quartz with 20 per cent 
peat 

Quartz with 50 per cent 
peat 

Peat 

Quartz with 20 per cent 
manure 



TREATMENT 



None 
Heated 

None 
Heated 

None 
Heated 

None 
Heated 

None 
Heated 

None 
Heated 



GERMINATION AFTER 



24 

hours 



per cent 
10 
28 

35 
4 

40 


34 


34 


30 

1 



42 
hours 



per cent 

58 
60 

62 
19 

74 
3 

63 


70 


62 
21 



66 
hours 



per cent 

72 
68 

77 
68 

79 

7 

68 


77 


66 
50 



90 
hours 



per cent 

75 
75 

81 
79 

81 
30 

73 
10 

79 


69 
64 



114 
hours 



per cent 



82 
53 

76 
32 

80 
1 

70 

71 



138 
hours 



per cent 



54 



47 



per cent 

75 
75 

81 
80 

82 
54 

76 

47 

80 
2 

70 

71 



TABLE 5 
Loss in weight of soils on heating to 250°C. and on ignition 



Peat 

Muck 

Waukesha silt loam 
Virgin sandy loam . . 
Fine sandy loam . . . 

Red clay 

Norfolk sand 



LOSS ON 

HEATING TO 

250°C. 



Per cent 
5.4 
1.4 
0.83 
0.76 
0.55 
0.43 
0.35 



LOSS 
ON IGNITION 



per cent 
68.1 
14.8 
5.4 
5.6 
4.3 
5.5 
1.9 



ORGANIC MATTER 

LOST ON 

HEATING TO 

250°C. 



per cent 

7.8 

9. 
15. 
13. 
12. 

7. 
18. 



sand and well decomposed peat were mixed and heated, and the toxicity was 
found to be in a large measure proportional to the amount of organic matter 
present. This, however, cannot be considered to approach conditions present 
in normal soils with varying percentages of organic matter. Table 5 shows 
the percentage loss of organic matter in soils heated to 250°C. as compared 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 13 

with the total organic matter present as measured by the loss on ignition to- 
gether with the per cent of the organic matter lost on heating to 250°C. Al- 
though the data at hand upon this subject are not sufficient to be significant, 
it is worth noting that the percentage of matter lost on ignition for the most 
toxic and the least toxic soil are too close to warrant any degree of importance 
being attached to them. It appears from these results, however, that peat 
and muck though highest in organic matter and losing the greatest weight 
on heating to 250°C. did not lose as large a percentage of their organic matter 
content as the others and were not as toxic as Waukesha silt loam which had 
considerable less organic matter but lost proportionately a larger amount on 
heating to 250°C. The indications are, therefore; that the amount of toxicity 
produced on heating is not proportional to its organic matter, but that it is 
rather proportional to the state of the organic matter or to the percentage 
which is actually decomposed but not taken care of by the soil itself through 
its powers of absorption. These probabilities will be considered more in 
detail under a study of the nature of the toxic substance. 

That results may be quite different when lower temperatures and different 
seeds are used, may be seen by referring to table 6. Heating to 50°C. stimu- 
lated cabbage seed germinations on most soils. Heating to 100°C. some- 
times stimulated and at other times retarded germination. Heating to 250°C. 
retarded germination in all cases; while heating to 500°C. brought the soil 
back practically to normal in all cases as far as toxicity is concerned. Heating 
to 800°C. either stimulated germination, or heating to such a high tempera- 
ture may be regarded as having merely eliminated the inhibiting elements 
naturally present in soils. The difference in results at the lower temperatures 
from that at higher temperatures has already been explained as being believed 
to be due to the stimulating action of a small amount of the toxic substance. 
This stimulating action may be considered to be exaggerated in table 6 on 
account of the use of cabbage seed in the germination test which is more "re- 
sistant" to the toxic substance than lettuce seed, as will be shown in the fol- 
lowing paragraphs. If wheat seed had been used the acceleration would 
have been more marked. The Waukesha silt loam soil being found to be 
most toxic to seed germination when heated, has been used most in the toxicity 
experiments. 

Influence of type of seed used on results secured. The great variation in 
susceptibility of seeds of different species of plants to the toxic action of heated 
soils opens two lines of interesting inquiry: the first being as to the nature of 
the toxic substance produced when the soil is heated, and the other being the 
causes of these differences in "susceptibility." The former is the one of most 
concern in the problems undertaken. The latter is a physiological problem 
often encountered in dealing with the relation of various chemicals to plant 
tissues, and may have an important bearing in explaining the nature of the 
toxic agents in heated soils. If a correlation could be established between 
variations in "susceptibility" of different seeds to heated soils with that of the 



14 



JAMES JOHNSON 



TABLE 6 

Efect of heating various soils to different temperatures on germination of cabbage seed 









GERMINATION 


AFTER 








HEATED 
TO 










Total 


SOIL 


48 
hours 


72 
hours 


96 

hours 


120 

hours 


144 
hours 




°c. 


per cent 


Per cent 


per cent 


per cent 


per cent 


per cent 




Check 


71 


79 


84 






84 




50 


76 


80 


82 


83 




84 


Peat* < 


100 


50 


80 


85 


88 




89 


250 


21 


32 


40 


53 


65 


77 




500 


85 


93 


95 






96 




800 


83 


86 


89 


93 




93 




Check 


77 


87 


90 


92 


93 


93 




50 


92 


94 


94 


95 




95 


Sparta sand < 


100 
250 


81 
40 


90 
60 


91 
66 


92 
68 


93 
70 


93 




80 




500 


73 


81 


86 


90 




90 




800 


86 


91 


91 


93 




94 




Check 


80 


88 


90 






90 




50 


73 


78 


81 


82 




83 


Red clay < 


100 
250 


79 
30 


89 
47 


89 

54 


91 

57 


92 
67 


92 




92 




500 


79 


84 


86 


88 


89 


89 




800 


79 


85 


87 


88 




91 




Check 


72 


87 


89 






90 




50 


74 


84 


88 






88 


Waukesha silt loam < 


100 
250 


7 
4 


17 
7 


24 
8 


31 
11 


32 
13 


83 




81 




500 


72 


79 


84 


85 




86 




800 


79 


83 


87 


90 




90 




Check 


71 


80 


80 






80 




50 


89 


92 


95 






97 


Fine sandy loam < 


100 
250 


91 
63 


96 

72 


98 
81 


99 
83 




99 




86 




500 


84 


87 


89 


90 




92 


. 


800 


79 


85 


88 


90 




90 




Check 


77 


87 


89 


91 




91 




50 


86 


89 


93 


94 




94 


Miami silt loam ■ 


100 

250 


63 
47 


86 
68 


92 

77 


93 
83 


94 
86 


96 




95 




500 


71 


78 


80 


81 


84 


85 




800 


S3 


87 


90 


90 


93 


93 



'An undecomposed peat, not the same as described in table 1. 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 15 

toxic action of certain pure chemicals on the assumption that seeds react 
differently to various chemicals, good indirect evidence of the nature of the 
toxic substance could be established. 

The reaction of seeds to chemicals or to heated soils may be either in the 
direction of stimulated or retarded rate of germination, or in increased or de- 
creased total percentage germination. The experiments are, however, con- 
fined to and based mostly upon the effect on rate of germination. If we select 
seeds of a wide range of families of cultivated plants as is shown in table 7 and 
compare the germination on soil heated to 250°C. with the germination on 
unheated soil, it will be found that on the particular soil used, the seeds of all 
families are retarded in germination. It can be noted, however, that the 
seeds differ very markedly in their resistance to the toxic property, as inde- 
cated by the relative germination at a certain period. The rapid recovery 
from the toxic action by some of the species and the final increased percentage 
of germination of certain species above that on unheated soils is notable. 
The seeds of wheat and cucumber are seen to belong to the very "resistant" 
class, whereas those of clover and lettuce are very ''susceptible" to the toxic 
property. Various gradations between the most and least susceptible species 
occur, and it is reasonable to suppose that other seeds more resistant and 
more susceptible than those tested here exist in nature. Working with a 
soil becoming very toxic on heating to the temperature most productive of 
toxicity (i.e., 250°C.), the advantage in simplifying the facts, over that on 
heating this soil to a lower temperature, is to be seen in table 8, where Wau- 
kesha silt loam was heated to 115°G. and sown to different seeds. In this 
case, we have no retardation but stimulatipn of the most resistant species 
together with marked retardation of the "susceptible" species, and it becomes 
easier to misconstrue the nature of the toxic properties present. 

Various factors of secondary importance influencing germination on heated soil. 
Turning to some of the other factors mentioned which deserve consideration 
as influencing or explaining the results noted above, it may be well to consider 
them briefly at this point, illustrating by data only those which may be es- 
pecially important or interesting. In all experiments on seed germination and 
plant growth the seed has usually been sown as soon as possible after heating 
the soil, this time rarely exceeding 6 hours, and where delayed has been usu- 
ally due to difficulty in wetting certain of the soils uniformly after heated to 
a low temperature. Naturally, however, the question arises as to what may 
happen in soils stored under different conditions for a considerable period of 
time between heating and seeding. All experiments of this nature have gone 
to show that the toxic property remains practically unchanged if the soil is 
kept in a dry condition, but that if the heated soil is kept in a moistened con- 
dition for a considerable period of time, changes occur and the toxic property 
is gradually lost or destroyed. This point is illustrated in table 9 where two 
different soils heated to 250°C. were kept for three months in the moist and dry 
condition, the toxicity as shown by seed germination being entirely lost in the 



TABLE 7 
Relative rate of germination of different seeds on soil heated to 250°C.; Waukesha silt loam 





SOIL 
IREATMENI 


GERMINATION AFTER 




SEED 


' 42 
hours 


66 
hours 


, 90 
hojrs 


114 
hours 


138 
hours 


162 
hours 


186 
hours 


TOTAL 






Per cent per cen 


( Per cen 


per cen 


per cen 


per cen 


per cent 


per cent 


Garden cress ' 


r Check 
Heated 


26 



34 
1 


44 
1 


62 
2 


67 

3 


68 

5 


15 


68 
24* 


Wheat 1 


' Check 
Heated 


36 
11 


76 
68 


86 
87 


93 
94 


95 






93 




96 


Lettuce i 


Check 
Heated 


69 



72 



73 

1 


83 
2 


85 
4 


88 
7 


27 


88 




50* 


Clover < 


Check 
Heated 


44 



68 
3 


79 
3 


85 
5 


86 

7 


9 


37 


86 




68* 


Flax 1 


Check 
Heated 


96 

21 


97 

57 


98 
71 


86 


90 


92 


95 


98 




95 


Cucumber I 


Check 
Heated 


41 
23 


65 
54 


75 
68 


84 
82 


85 
84 


86 

85 


87 
87 


88 




88 


Cabbage I 


Check 
Heated 


3i 



53 



59 
1 


65 

3 


65 
11 


66 
29 


51 


66 




56* 


Onion i 


Check 
Heated 


3 



16 

1 


45 
8 


73 
54 


78 
70 


78 
73 


80 

75 


80 




75 


Spinach i 


Check 
Heated 




5 



15 
1 


37 
5 


48 
13 


51 
20 


57 
36 


59* 

47* 


Buckwheat I 


Check 
Heated 






8 
1 


20 
12 


29 

22 


46 

37 


61 
60 


63 
64 


Carrot | 


Check 
Heated 






43 



59 

2 


64 
15 


67 

42 


63 


67 
68 


Tomato i 


Check 
Heated 








30 



54 
1 


58 
10 


65 

53 


67 
61 


Phlox 1 


Check 
Heated 








7 



11 



13 



20 

3^ 


24 
11* 


Snapdragon < 


Check 
Heated 








6 



31 



41 



47 
1 


49 

8* 


Cotton 1 


Check 
Heated 








5 



14 

1 


39 
11 


72 
65 


74 
73 


Linaria < 


Check 
Heated 










17 



39 



58 



67* 

2* 


Parsnip I 


Check 
Heated 












4 



12 
4 


17* 

8* 


* Seeds moldy and 
to this 


failure to 


show a 


greatei 


total germination m 


ay be partiallj 


' due 








16 















INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



17 



moist state but quite largely maintained in the dry state. This persistence 
of the toxic agent is further shown in table 10 where heated soil kept dry for 
two years still showed a very considerable toxic action. That aeration has 
no particular influence in this regard where soil is stored dry but that it has 

TABLE 8 
Relative rate of germination of different seeds on heated soil. Waukesha silt loam heated to 115°C. 









GERMINATION AFTER 








SOIL 
TREATMENT 
















42 
hours 


66 

hours 


90 
hours 


114 
hours 


138 
hours 


258 
hours 








per cent 


per cent 


Per cent 


per cent 


per cent 


percent 


per cent 


Clover I 


None 


65 


81 


85 


86 


88 




88 


Heated 


57 


74 


80 


84 


87 


88 


88 


Cabbage \ 


None 


56 


83 


88 


90 


91 


92 


92 


Heated 


69 


82 


87 


88 


89 


90 


91 


Garden cress s 


None 
Heated 


20 
16 


48 
40 


64 

52 


68 
56 


71 
60 


72 
66 


73 




66 


Onion \ 


None 


2 


67 


93 


95 


96 


97 


97 


Heated 


4 


64 


93 


96 


97 




97 


Buckwheat \ 


None 
Heated 




7 
3 


14 
14 


23 
21 


29 


55 
42 


55 
42 


Tomato i 


None 




9 


84 


96 


97 


98 


98 


Heated 




2 


69 


91 


98 


99 


99 


Carrot i 


None 




10 


36 


53 


58 


64 


64 


Heated 


• 


3 


24 


50 


62 


67 


67 


Lettuce <, 


None 
Heated 


83 
8 


92 
40 


94 
67 


83 






94 

83 


Flax I 


None 


97 


98 










98 


Heated 


92 


94 


95 








95 


Wheat 1 


None 


30 


93 


97 








97 


Heated 


47 


96 


97 








97 


Cucumber s 


None 
Heated 


5 
10 


65 

71 


82 
86 


84 
87 


85 
88 




86 . 
88 



some influence where soil is stored moist, is shown in table 11, in which ex- 
periment, soils were stored in Mason jars with covers sealed on in one case, 
but left off in others. No consideration was given to reinfestation with 
microorganisms in this experiment. This test indicates that the time of 
seeding following sterilization or heating has little influence if the soil is kept 



son, SCIENCE, VOL. VII, NO. 1 



18 



JAMES JOHNSON 



TABLE 9 

Efed of storage of healed soil for 3 vipnlhs in moist and dry condition upon rate of seed 
germination; cabbage seed 











GERMINATION AFTER 








TREATMENT 


STORED 












TOTAL 




43 hours 


55 hours 


67 hours 


79 hours 


97 hours 










per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


Waukesha silt 


Check 1 


Dry 

Moist 


49 

53 


53 
65 


70 
70 


73 
74 


73 
76 


75 
78 


loam 


Heated to 250^C. I 


Dry 

Moist 


13 
50 


25 
61 


38 
66 


50 
71 


60 

73 


72 
75 




Check 1 


Dry 


43 


53 


62 


66 


66 


66 


Fine sandy 


Moist 


45 


55 


62 


64 


66 


68 


loam 


Heated to 250°C. | 


Dry 

Moist 


19 
49 


39 
65 


50 
68 


61 

73 


64 
76 


70 

78 



TABLE 10 



Influence of two years storage of dry heated soil on relative rate of seed germination; 
Waukesha silt loam; clover seed 



SOIL TREATMENT 


STORED DRY 


GERMINATION AFTER (pERIOD) 


TOTAL 




1 


2 


3 


4 


5 


6 




Not heated 





2 years 
2 years 


Per cent 
41 


20 




per cent 

80 

2 

75 
11 


per cent 

86 
4 

81 
22 


per cent 

7 
83 
42 


per cent 
11 

85 
66 


per cent 

14 
87 
70 


per cent 
86 


Heated to 250°C 


53 


Not heated 


87 


Heated to 250°C 


75 







TABLE 11 

Influence of moisture and aeration of heated soil during storage on germination of seed; 
Waukesha silt loam; lettuce seed 





STORAGE 




GERMINATION AFTER 






TREATMENT 












TOTAL 




Water 


Air 


24 hours 


36 hours 


48 hours 


60 hours 


72 hours 


84 hours 










Per cent 


per cent 


per cent 


per cent 


per cent 


Per cent 


per cent 


' 


-{ 


+ 


75 


95 


96 








96 




— 


26 


87 


91 


92 






92 


Not heated ] 


J 


+ 


3 


62 


89 


92 










92 




I 


— 


11 


76 


87 


91 






91 


r 


M 


+ 





10 


58 


73 


74 


74 






— 





2 


19 


48 


51 


52 




Heated to 250°C ] 






















/ 


+ 





1 


1 


2 


8 


9 






I 


— 











2 


4 


5 





INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



19 



dry, but that the toxicity is gradually lost where heated soils are kept moist 
and exposed to aeration and contamination. It points further to the fact 
that some of the toxicity to seed germination may be diminished toward the 
close of a seed germination test in Petri dishes, and also that a part of this, 
which if given off from the soil rather than destroyed in it, may be partially re- 
tained in the enclosed chamber due to lack of good aeration, and hence ex- 
aggerate the retardation which fact may partly explain the more marked 
retardation under these conditions than is ordinarily observed on heated 
soils under natural conditions. 

That the amount of moisture present in the soil at the time of heating may 
influence the amount of toxic substance formed or its retention by the soil is 
shown in table 12. Here 50 gm. of Waukesha silt loam were placed in Petri 
dishes and varying amounts of water added before heating in the autoclave, 
after which it was made up to equal percentages of water and seeded to lettuce. 

TABLE 12 

Relative rate of germmation of seed on soil heated with different amounts of moisture; 
Waukesha silt loam; lettuce seed 



TREATMENT 


WATER 

ADDED TO 

50 CMS. 


GERMINATION AFTER 


TOTAL 


















AIR-DRY SOIL 


30 hours 


42 hours 


54 hours 


66 hours 


78 hours 






CC. 


per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


Not heated 





76 
11 


89 
29 


91 
68 


94 

87 


95 
89 


96 




93 




5 


17 


49 


79 


89 


92 


92 


Heated 115°C < 


10 
15 


10 
6 


27 
23 


71 
63 


87 
82 


91 
85 


92 




89 




20 


4 


19 


61 


80 


87 


94 




25* 


2 


16 


52 


80 


85 


90 



* Saturated. 

Increased water content at the time of sterilization apparently increases the 
toxic action. There is also, however, an indication that adding a small percent- 
age of water may be less productive of injurious products than heating an air 
dry soil, although this may be associated with physical changes, since the ability 
of relatively dry soil heated to 115°C. or thereabouts, to absorb moisture is 
very poor. Throughout the experiments, the soils used for heating have been 
usually air dried, this of course, being advantageous in heating to higher tem- 
peratures with dry heat. 

As to time of heating, an arbitrary standard must be chosen and maintained 
as closely as possible, the soil being allowed to become gradually and as uni- 
formly heated to the desired temperature as possible within that period of time. 
Where large quantities of soil are heated it is important to mix the heated 
soil thoroughly before taking fractions for tests. To determine the effect of 
length of time of heating a soil on seed germination, the Waukesha silt loam 
was heated for different lengths of time, ranging from 10 to 160 minutes, in the 



20 



JAMES JOHNSON 



autoclave at 115°C. (table 29). The soils were spread out in a thin layer to 
make thorough heating at the short periods of time possible. Ammonia de- 
terminations and freezing point determinations of the soil solution were also 
made in this experiment. The differences due to length of time of heating 
at this temperature were small and perhaps insignificant, but there is seem- 
ingly a slight increase on increased time of heating followed by a lowering of 
toxicity to seed germination which then remains fairly constant and although 
changes do occur they do not seem to be constantly in one direction. This 
was seemingly not correlated with ammonia content or concentration of the 
soil solution. Similar rise and fall of toxicity was also obtained for soil heated 
to 200°C. for 2, 4, 6, 8, 10, and 12 hours, but no decisive conclusion could be 
drawn as to the influence of time of heating on seed germination. 

TABLE 13 

Efed of temperature of germination on toxic actipn of heated soil; Waukesha silt loam; red clover 





TEMPERA- 




GERMINATION AFTER 








TURE OF 
GERMINA- 
TION 










son. TREATMENT 


48 
hours 


72 

hours 


96 
hours 


120 
hours 


144 
hours 


TOTAL 




°C. 


per cent 


Per cent 


per cent 


per cent 


per cent 


per cent 




12 


5 


40 


68 


80 


81 


82 




15 


14 


52 


67 


73 


76 


79 


None < 


21 

24 


39 

77 


79 

88 


90 
91 


91 
91 


92 
91 


92 
91 




26 


75 


86 


86 


87 


87 


87 


. 


• 35 


27 


37 


44 


56 


60 




f 


12 








2 


9 


19 


77 




15 





1 


5 


10 


19 


85 


Heated to 250°C < 


21 

24 



6 


5 
15 


12 
21 


20 
40 


25 
67 


83 




71 




26 


2 


3 


5 


27 


58 


60 




35 














1 





To determine the relation of the temperature at which the germination 
test is made upon the degree of toxicity shown by heated soils, the results 
shown in table 13 were secured. Waukesha silt loam soil was used, and the 
heated and unheated checks were placed at different temperatures in a series 
of Altmann and other electrically regulated incubators where constant tem- 
peratures could be maintained. The optimum temperature for the germi- 
nation of clover seed was found to be close to 24-26°C. The same tempera- 
ture apparently held for germination in both heated and unheated soils. 
The proportion of germination seemingly holds the same for all temperatures 
on both heated and unheated soils excepting at the highest temperature, i.e., 
35°C. where the injurious action of the combination of high temperature and 
soil toxicity is proportionally greater for the heated soil. It is apparent, 
however, that a fairly large range of temperature variations will not materi- 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



21 



ally effect results in the germination experiments. No special efforts were, 
therefore, made to keep all germination tests at constant temperatures, and 
usually only room temperatures (about 20-22°C.) were used. These were 
kept quite constant day and night, however, by the automatic regulation of 
the steam-heating system. 

In the preceding temperature test of germination, lettuce seed was used in 
the first trial. It was found that the lettuce seed germinated very poorly in 
all the dishes in the incubators which excluded light. It was at first thought 
that poor seed had been used but seed from the same samples in the laboratory 
germinated normally. It was, therefore, concluded that light must be a 
controlling factor in lettuce seed germination, and this was found to be the 
case. It has, of course, been repeatedly shown that light is necessary for 
normal germination in many seeds (38). As lettuce seed was most commonly 

TABLE 14 

Influence of absence of light on the germination of lettuce seed in heated and unheated soil; 

Waukesha silt loam 







GERMINATION AFTER 




SOIL TREATMENT 


LIGHT 


30 

hours 


48 
hours 


72 
hours 


,96 
hours 


120 
hours 


TOTAL 






Per cent 


per cent 


per cent 


per cent 


Per cent 


per cent 


None < 


4- 


75 
1 


88 
7 


90 

8 


93 
9 


95 
10 


97 
10 


Heated to 115°C < 


+ 


12 
3 


40 

4 


62 

5 


74 
5 


84 
6 


90 




8 


None I 


+ 


82 


93 


93 


94 


95 


96 


- 





10 


16 


20 


21 


22 


Heated to 250°C < 


+ 






2 
3 


8 
7 


15 
8 


23 
12 


26 




13 



used in the tests in my experiments, however, on account of their suscepti- 
bility to the toxic action of heated soils and their ease and convenience in 
handling, it was found necessary to keep the seeds germinating in the open 
room rather than in a dark incubator. An interesting fact noted in this 
connection (table 14) was that the combined action of absence of light and of 
soil toxicity did not retard seed germination in proportion to the expectations 
when compared with unheated soils in the absence of light. In other words, 
the heated soils apparently supplied in some small measure a substitute for 
light in germination. This is especially interesting considering the results of 
Gassner's work (20) in replacing the effect of Ught with certain nitrogenous 
salts. 

In an effort to determine the relation of size of soil particles used to ger- 
mination on heated soils, a heated sample was screened to different sized 



22 JAMES JOHNSON 

particles and sowed to lettuce. No particular conclusion is warranted from 
this test; although there is an indication that the larger-sized particles are 
less toxic than the finer grades. It is possible, however, that other factors 
than toxicity play a part here. The results are in agreement with those of 
Stone and Monahan (70) on this subject, and as suggested by them the pre- 
caution should be taken of using soils of a standard degree of fineness for 
germination tests. The addition of ammonia to soils of coarse and fine tex- 
tures indicated more markedly that greater toxicity occurred in soils of fine 
than in those of coarse particles. 

Pickering (50) has shown that moisture content of soil in germination tests 
may vary quite widely without greatly influencing the results. This is in 
accordance with results obtained in an experiment carried on by the writer 
in this connection. Too low moisture content reduces, of course, the rate of 
germination, while the higher moisture contents tend to increase rate of 
germination, but probably reduce total germination shghtly. It has already 
been mentioned that certain soils heated to low temperatures take up water 
with great difficulty. The loss of capillarity is in some instances very strik- 
ing, the soil seeming in fact at times to have lost all affinity for water. In 
"kneading" the soil up with water in order to get some degree of uniformity 
of moisture throughout, it is possible that a physical condition is obtained 
unfavorable to germination quite apart from toxic action. This influence, 
however, has not been deemed sufficiently important in this connection to war- 
rant detailed study. Soils heated to high temperatures do take up water 
very much more readily than unheated soils, and it was found, for instance, 
that a virgin sandy loam heated to 250°C. took up water two to three times 
as fast as the unheated, while this soil heated to 115°C. took up water only 
one-half to one-eighth as fast as the unheated soil. 

The size of the seed is not considered as influencing the susceptibility to 
the toxic action of the heated soil. Corn and peas, for instance, being larger 
than rye or buckwheat, are more susceptible, while clover and lettuce being 
smaller than the latter, were also more susceptible. Similarly the gross chem- 
ical composition, as for instance starchiness and oiliness, does not seem to be 
in any way connected with susceptibility or resistance to injury. Sweet 
corn, for example, is only slightly more susceptible than field corn, and flax 
and wheat are about equally resistant. Age, or maturity of the seed, as far 
as noted does not apparently influence results to any extent. It seems most 
probable that the factor which determines the nature of the response in seed 
germination lies in the selective permeability of the seed coats to certain 
chemical substances. With the above facts in mind tests were begun with 
the idea of determining and measuring more closely the nature of the toxic 
substances in heated soils. Before taking this up it is probably advisable to 
consider how far these facts concerning seed germination are correlated with 
plant growth on heated soils. 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 23 

Plant growth on heated soils 

Although the beneficial action of heated soils on the growth of the higher 
plants had been known for a long time, it was apparently not until about the 
middle of the last century that any serious experimental investigation was 
undertaken in this field. Struckman (73) in 1857 in addition to other lines 
of work on burned soils obtained good results with cabbage and rape, but 
noted no especially good results with vetch and flax on burned soil. The 
experiments of Fleischer (17) and his associates at the " Moor-Versuchs 
Stationen" were continued for a number of years, heated soils giving on the 
whole good results with plant growth, although the economy of burning was 
open to question. With respect to the influence of dry heat on soil, the more 
recent investigations along this line are especially those of Pickering (53), 
Seaver and Clark- (68), Fletcher (18), and Kelley and McGeorge (30). A 
more extensive amount of literature exists upon plant growth in steamed 
soils, and there is a great deal of evidence showing the beneficial action of 
steamed soils on plant growth since the time of Franke (19), Pfeiffer and 
Franke (49), and Kruger and Schneidewind (37). Studies on the injurious 
action of heated soils began especially with the results of Deitrich (13), and 
these have since become a very important part of the investigations upon the 
subject. Following the work of Girard (21) who appUed carbon bisulfide 
to the soil used for the growth of several agricultural crops, a large amount 
of literature has accumulated with the object of explaining the beneficial 
action on plant growth of antiseptics applied to soils. That sterilization by 
chemical agents and by heat have one and the same effect on soil as far as 
plant growth is concerned has been generally accepted, and the two methods, 
sterilization, or "partial sterihzation" by heat or chemicals are, therefore, 
from a biological standpoint, usually considered together by most authors. 
The theories concerning the causes of the modification of plant growth on 
sterilized soils have already been briefly mentioned, and the changes in and 
caused by sterihzed soils outlined. The experimental results presented here 
are mainly concerned with establishing further evidence relative to the toxic 
and beneficial action of heated soils, and the immediate and subsequent 
changes produced on different soils as measured by the growth of different 
plants, with the purpose of arriving at a better conception of the nature of 
the beneficial and toxic substances produced in heated soils. Experiments 
were therefore undertaken to determine the influence of the following as 
measured by plant growth: (a) the response of different types of soil to ster- 
ilization; (b) the effect of different temperatures of heating; (c) the effect 
on different kinds of plants; (d) the influence of different lengths of time of 
heating; (e) the influence of repeated heating; (f) the influence of various 
soil environmental factors on the expression of the injurious and beneficial 
action. 



24 JAMES JOHNSON 

Soil type. The remarkable variation in results which may be secured with 
plant growth on different soils when sterihzed is best illustrated by table 15. 
As wide a variety of soil substances as could be secured were used including 
those previously described, and also pure leaf mold, greenhouse compost, 
and a very acid sand (lime requirement 9.38 tons per acre). After being 
sterilized at about 110°C. these soils together with their checks were planted 
to tomatoes. A wide variation in results was noted from the start, both in 
quantitative and "qualitative" response, and these variations and changes 
continued to occur throughout the period the plants were allowed to grow. 
Comparing the yield of the different unsterilized soils it may be noted that 
the compost was apparently the most fertile and the red clay the least fertile 
for tomatoes. The greatest percentage increase in yield, however, occurred on 
the muck soil 275 per cent and the greatest reduced yield on the virgin sandy 
loam where the plants were practically all killed (plate 1, £g. 1). There was 
manifestly no correlation between the fertility of the soil and the toxic or 
beneficial action. Considering total organic matter, in those soils in which 
the loss by ignition has been determined (table 1) it may be seen that 
peat, highest in organic matter (68.1 per cent), though very toxic on 
being sterilized, was not so much so as virgin sandy loam with only approxi- 
mately 5.6 per cent organic matter. Muck with about 14.8 per cent organic 
matter was only slightly more beneficially influenced than Miami silt loam 
with only about 5.4 per cent organic matter. There is, therefore, also ap- 
parently a lack of correlation between the behaviour of the heated soils to- 
ward plant growth and their content of organic matter. That fertility and 
organic matter bear a close relation to the action on plant growth upon ster- 
ilizing the soil, has been repeatedly suggested by various writers but fre- 
quently disproven when subjected to experimental test. That the organic 
matter should bear some relation to the toxic and beneficial action upon heated 
soils is, however, too evident in connection with other facts to dismiss the 
matter without further consideration. The influence of the organic matter 
may be indirect and complicated by various factors, especially by its rela- 
tion to the absorptive capacity of the soil, its chemical state as influencing ex- 
tent of decomposition upon heating, and by its relation to bacterial activity 
subsequent to sterilization. The relation of absorptive capacity, especially, 
will be considered in connection with the discussion of the nature of the toxic 
agent. For the present we can only conclude that very little is suggested by 
the gross character of the soil itself as to its action upon plant growth when 
subjected to sterilization by heat. 

Aside from simple retardation and acceleration of growth, several other in- 
teresting results were obtained where tomatoes were grown in different soils 
heated to about 110°C. These are indicated briefly in table 15 but will be 
discussed in more detail under the subject of the "qualitative" action of 
heated soils. The extreme toxicity of virgin sandy loam to tomatoes when 
sterilized should not, however, be allowed to confuse the problem being pri- 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



25 



TABLE 15 
Air-dry weight of tomato vines* grown on different soils sterilized at 110°C. 



Miami silt 
loam 

Muck < 

Fine sandy / 
loam \ 

Norfolk sand . I 
Leaf mold ^ 



Red clay ^ 

Peat I 

Waukesha silt / 
loam \ 



Acid sand . 



Greenhouse 
compost . . . 



Virgin sandy / 
loam \ 



TREATMENT 



None 
Sterilized 



None 
Sterilized 

None 
Sterilized 

None 
Sterilized 

None 
Sterilized 



None 
Sterilized 



None 
Sterilized 



None 
Sterilized 



None 
Sterilized 



None 
Sterilized 



None 
Sterilized 



DRY 
WEIGHT 



gm. 
1.60 
5.65 



1.60 
6.00 

1.45 
3.05 

0.50 
0.90 

6.20 
6.95 



0.15 
0.15 



0.90 
0.30 



4.60 
2.10 



0.85 
0.35 



9.65 

5.35 



5.60 
0.15 



S + t 



l; R -^ 
Z w w 



oi « W ' 



EARLY GROWTH 



+254 Leaves mottled; slight 
retardation 



+275 



+110 



+ 80 



+ 12 



-200 



-119 



■142 



80 



No retardation 



Slight retardation 



Purple pigmentation 

Purple pigmentation 
Leaves mottled; slight 
retardation 

Purple pigmentation 
Purple pigmentation; 
no retardation 



Marked retardation; 
purple pigmentation 



Uneven retardation 



Purple pigmentation 
Purple pigmentation: 
slight retardation 



Leaves mottled ; slight 
retardation 



LATE GROWTH 



Leaves yellowing 
Leaves a healthy green 



Leaves yellowing 
Leaves a healthy green 



Change in pigmenta- 
tion and recovery 
started 



Marked toxic action 
on some 



Several spots and le- 
sions on leaves and 
stems 



Toxic action started 
on leaves 



Marked retardation; Plants practically 
lesions on leaves killed by lesions on 

leaves and stems 



* Six plants — 62 days' growth in duplicate pots. 



26 JAMES JOHNSON 

marily considered, i.e., simple retardation. This extreme toxic action may 
not be due to the same cause as that of simple retardation. The percentage 
decrease on the virgin sandy loam is not included in the data given. This 
type of injury in which plant tissue is actually destroyed, has been termed 
"chemical" injury in order to distinguish it from retardation where no such 
injury appears to occur. That "chemical" injury is not confined to vir- 
gin sandy loam soil alone, however, is evidenced by the same but less marked 
occurrences in three of the other soils used. The results secured in table 15 
would have been quite different if other plants had been used, as will be 
shown later. On the other hand, tomatoes are not the only plants subject to 
forms of "chemical" injury. Furthermore the temperature to which the 
soil is heated, the length of time of heating, and the temperature of the soil 
during the growing period are all influential in determining the occurrence and 
extent of injury resulting. The results presented in table 15 only show, 
therefore, what may occur under a certain set of conditions when various 
types of soil are used. It should also be noted that the results as shown 
by the final yields are only comparative for that stage of plant growth 
at which the crops were harvested. It requires a longer period for some 
soils or plants to recover from the toxic action than others, and in 
certain instances the greater the toxic action and the time required 
for recovery, the more pronounced the subsequent increase. The yields 
should preferably be taken therefore at the maturity of the plants. In 
experiments under greenhouse conditions this is frequently impossible with 
certain crops and is not regarded as essential with others. The yields given 
are therefore in terms of dry vegetative matter produced during a certain 
period of time regardless of maturity but usually at a stage when conditions 
similar to that which may be expected at maturity have been reached. Per- 
centages of increase or decrease in yields, therefore, may not indicate the 
maximum of either but rather the comparative yields at a certain time. On 
the other hand it is possible so to regulate the time of taking the yield, by 
noting the stage and vigor of recent growth, that any serious error in this 
respect may be avoided. 

In connection with the studies on influence of soil type, an experiment was 
conducted to determine whether any difference existed between surface soil 
and subsoil in their behavior toward sterilization. Stone and Monahan (70) 
convey the impression that such a difference exists owing to the location of 
the soils rather than to their chemical or physical characteristics, although 
it is probable that the authors did not intend to convey such an impression. 
These writers report a decrease in yield of soy beans in sterilized subsoil as 
compared with sterilized surface soil. These results could not be corrobo- 
rated. With soy beans a Miami silt loam surface soil showed an increase of 
91.3 per cent and the subsoil an increase of 118.7 per cent over the unsterilized 
soil. Virgin sandy loam surface soil showed a decrease of 20 per cent for the 
surface soil and an increase of 58.3 per cent for the subsoil. These tests 



INFLUENCE OF HEATED SOILS ON CERMLNATION AND GROWTH 



27 



though not carried out in detail, indicate that nothing definite can be con- 
cluded from the location of the soil with reference to the surface as to its ac- 
tion to plant growth upon sterilization. The results are, on the other hand, 
not correlated with content of organic matter in the soil as suggested by Stone 
and Monahan's conclusions. 

Temperature of heating. The temperature to which a soil is heated has a 
marked influence upon its behavior toward plant growth as is illustrated by a 
preliminary experiment (table 16) where cabbage was used on two different 
soils heated to 115°, 250°, and 350°C. The early plant growth was retarded 
to the greatest extent on both soils on heating to 250°C., but the subsequent 
beneficial action is seen to be the greatest at this temperature of heating. The 
muck soil gave a decidedly greater beneficial action on heating than did the 
Waukesha silt loam, but it is not to be expected that all soils will show bene- 
ficial action at a given time after heating to 250°C., nor that all plants will 

TABLE 16 
Dry weight of cabbage produced in soil on heating to different temperatures 



TEMPERATURE OF 


MUCK 


SOIL 


WAUKESHA SILT LOAM 














Weight 


Increase 


Weight 


Increase. 


"C. 


gm. 


per cent 


gm. 


Per cent 


Check 


1.95 




2.55 




115 


8.60 


340.7 


3.40 


ii.z 


250 


9.70 


402.5 


4.95 


94.1 


350 


4.35 


123.0 


3.60 


41.1 



respond in the same manner. On heating six different soils to 250°C. the 
increase in yield of tobacco, for example, ranged as follows: Muck, 571 per 
cent; Waukesha silt loam, 473 per cent; clay, 150 per cent; fine sandy loam, 
96 per cent; virgin sandy loam, 62 per cent, while the peat soil showed a de- 
crease of 25 per cent. The retarding influence of the peat, however, would 
have been represented by a much greater figure earlier in the test, while, on 
the other hand, at the time of cutting the plants they were growing vigor- 
ously, indicating that in two or three weeks the yield on the heated peat 
would have exceeded that on most of the other heated soils. Such seeming 
discrepancies in results do not alter the fact, however, that there exists a 
maximum degree of toxicity followed usually by a maximum degree of bene- 
ficial action to plant growth on soils heated to approximately 250°C. That 
this critical temperature lies closer to 250°C. than to 200° or 300°C. has been 
repeatedly noted and is illustrated in table 17 where the relative growth at 
the end of one month (placing the largest growth at 100), together with the 
final yield is shown. In a similar manner the retarding action of virgin sandy 
loam to tomatoes is shown in plate 1, figure 2 and the benefits of heated muck 
soil on radish is shown in plate 1, figure 3. The decreased yield (table 17) 



28 



JAMES JOHNSON 



on heating to 50°C. is believed to be due to loss of nitrates or other forms 
of nitrogen on drying, together with no beneficial action in the way of in- 
creased availability of plant food, due to heating at this low temperature. 
While tobacco recovered relatively rapidly from the toxic action, other plants 
may require a relatively much longer time (plate 2, fig. 1), and may even in 
some instances entirely fail to recover during the normal life of one annual 
plant, although such "recovery" of the soil may be shown in the later crops. 
The time required for recovery is, usually, in proportion to the degree of 
toxicity; consequently we find that on soils heated to different temperatures 
the earliest growth is usually best on those soils not heated or on soils heated 
to 350°C. or above, where no toxic principle is present. If the toxicity at 
100°C. is relatively small a rapid recovery occurs and in the space of two 
weeks to a month it will usually be found that the plants on soils heated to 
this temperature are most vigorous owing to the beneficial action obtained. 



TABLE 17 
Influence of healing soil to different temperatures on yield of tobacco; Waukesha silt loam 



TEMPERATUKE OF 
HEATING 



Check 
50 
100 
150 
200 
250 
300 
350 



RELATIVE GROWTH AT END 
OF MONTH (estimated) 



95 

60 

100 

90 

85 
75 
85 
95 



DRY WEIGHT 


INCREASE 


gm. 


per cent 


1.55 




1.15 


-34 


2.70 


74 


4.85 


212 


5.20 


235 


6.40 


312 


5.90 


280 


4.05 


161 



At this time, however, the plant growth at the 250°C. temperature will usually 
be the poorest with less retarding action at the 200° and 300° temperatures. 
Recovery then follows at 150°C., 300°C., 200°C., and finally at 250°C. Once 
the injurious action is lost, the increase in plant growth occurs relatively rap- 
idly and at a time when the soils heated to the lower temperatures seem to 
have exhausted the beneficial property produced, the soils heated to 200° 
and 250°C., may be at their height of vigor of growth. The apparent 
correlation between the action of soils heated to different temperatures on 
plant growth and their action on seed germination is evident, and seem- 
ingly indicates that the toxic principle is the same in both cases. 

Results vary with kind of plant grown. The extreme toxicity of heated 
virgin sandy loam soil to tomatoes has been shown. If now, different types 
of plants are grown in this soil, heated to the same temperatures (table 18), 
it i^ found that certain plants, in fact most plants, thrive on this heated soil 
which is so toxic to tomatoes as to practically kill them in many instances 
(plate 2, fig. 2). Buckwheat and wheat show an increased growth of 321.8 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



29 



per cent and 245.9 per cent, respectively, while tomatoes show a decrease of 
41.7 per cent (table 18). This experiment was conducted during the summer 
and the decrease is considerably less than that which may be expected to oc- 
cur during cooler seasons of the year, as will be shown later. That this differ- 
ence in "resistance" and "susceptibility" of plants to the toxic action of 
heated soils is characteristic on all soils and with all plants cannot be doubted 
although no attempt has been made to prove this point. The results ob- 
tained at any one time are always complicated by a considerable number of 
factors, of which probably the most important is environment, and conse- 
quently the results are likely to be confusing unless properly analyzed. The 



TABLE 18 
Effect of sterilization of soil at 115°C., in autoclave on yield of different crops; virgin sandy loam 



Buckwheat . 



Radish . 



Soy beans . 



Wheat. 



Lettuce. 



Tomatoes . 



TREATMENT 


DRY 
WEIGHT 


IN- 
CREASE 


REMARKS 




gm. 


per cent 




None 


3.20 






Heated 


13.50 


321.8 


Early growth markedly retarded 


None 


2.15 






Heated 


8.45 


293.0 


Early growth retarded 


None 


8.10 






Heated 


8.05 





Early growth retarded. Foliage 
shows "chemical" injury 


None 


1.85 






Heated 


6.50 


245.9 


Early grow retarded 


None 


0.80 






Heated 


1.60 


100.0 


Early growth retarded 


None 


1.15 






Heated 


0.67 


-41.7 


Early growth retarded. Marked 
"chemical" injury 



results of one other experiment may be cited, however, in this connection in 
which three different soils heated to 100°C. were grown to four different crops. 
Muck soil gave an increase of 140.9 per cent for radish as compared with 40.6 
per cent for lettuce. Heated fine sandy loam increased the yield of wheat 
119.1 per cent, but decreased the yield of lettuce 106.6 per cent, and virgin 
sandy loam increased the yield of radish 1.7 per cent as compared with an 
increase in this case of 76.4 per cent for tomatoes. These results illustrate 
further the marked variation in the response of plants to sterilized soils. Other 
results of this nature have shown in general that the Gramineae as a whole 
are relatively resistant to the toxic action and consequently show relatively 
high beneficial results from heated soils. The Solanaceae and Leguminoseae 



30 JAMES JOHNSON 

are on the other hand apparently more susceptible to the toxic action, although 
great variation exists within the families in this respect. This seems roughly 
to correspond with the action on seed germination, but experiments to be 
referred to later along this line apparently fail to bring out any decided 
correlation. 

Length of time oj heating. The work of Seaver and Clark (68) and of 
Pickering (50) have indicated that the length of time the soil is held at a 
certain temperature does not materially influence its behavior toward plant 
growth. Some preliminary experiments on this aspect of the problem tended 
to corroborate their conclusions, but on the other hand there appear to be too 
many exceptions to permit of applying this conclusion as a general rule. Fine 
sandy loam soil heated to 115°C. for various lengths of time ranging from 10 
to 180 minutes and planting to wheat showed that a gradual increase in yield 
occurred up to 120 minutes of heating but fell off again slightly on heating to 
180 minutes. In this case it should be noted that a soil producing only a 

TABLE 19 

Influence of length of time of heating virgin sandy loam at 115°C. on growth of tomatoes 



TIME OP HEATING 


DRY WEIGHT AVERAGE TRIPUCATES 


minutes 


gm. 


Check not heated 


2.28 


10 


5.57 


20 


4.77 


40 


2.63 


80 


0.50 


160 


0.10 



slight toxic action on heating and also a plant resistant to this toxic 
property were used. When tomatoes were grown on muck soil a different 
result was secured. Heating 10 minutes gave an increase in yield of 132 
per cent, 20 minutes an increase of only 53 per cent which on heating 80 
minutes was again raised to 157 per cent, but again fell off to 118 per cent 
on heating 160 minutes (plate 3, fig. 1). This type of behavior both with 
seed germination and plant growth has, however, been observed too fre- 
quently to dismiss as insignificant. The behavior seems to indicate that 
the balance between the toxic and beneficial action is altered by the length of 
time of heating, probably due to the altered balance between the production 
and the volatilization of one or more products from the soil on heating. More 
definite evidence has been secured, however, to show that the length of time 
of heating may have a marked action on the behavior toward plant growth. 
Virgin sandy loam was heated for various lengths of time between 10 and 
160 minutes at a temperature of 115°C. and set to tomatoes (plate 3, fig. 2). 
The toxic action as shown by "chemical" injury :first appeared in the soil 
heated for 160 minutes, and appeared successively in the other soils to and 
including 20 minutes of heating. At 10 minutes an almost immediate bene- 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 31 

ficial action was found and persisted throughout the experiment. The 
soils heated for 20 and 40 minutes recovered in considerable measure and 
made a better growth than the checks, but they still showed the toxic action 
on the lower leaves at intervals. The soils heated for 80 and 160 minutes 
gave practically no growth of tomatoes, the leaves dying off almost as 
rapidly as formed. The average yield from triplicate pots are shown in table 
19. It is at least very evident that the length of time of heating certain soils 
does influence the growth of certain plants. 

Repeated heating of soils. Practical plant culturists using sterilization 
by heat often inquire as to whether soils once sterilized may again be steri- 
lized and replanted with the assurance of results as good as produced by 
the first sterilization. Some experimental evidence has been secured on this 
point in two different ways: first by repeatedly sterilizing a soil after each 
successive crop, and second, by sterilizing a soil on successive days before 
planting. The results of the first tests, in which fine sandy loam was used 
and grown to lettuce, indicated that the soil responded in a beneficial way 
after each sterilization in very much the same way as previously unsterilized 
soils did. In another experiment in which wheat was used on muck soil and 
on virgin sandy loam, and the same soil was heated 1, 2, 4, and 8 times on 
successive days to 115°C. for 90 minutes, no marked difference in results was 
obtained, a beneficial action being exhibited in all cases above the unheated 
check. Some indication, however, that one heating was more beneficial (or 
less productive of toxic action) than repeated heating, is indicated by the 
figures (table 20). Another experiment conducted in essentially the same 
manner, vfsing greenhouse compost grown to tomatoes, again indicated that 
repeated heating on successive days increased the toxicity to tomatoes, al- 
though all the heated soils eventually recovered and produced final yields 
greater than the unheated checks (table 20). It does not follow, however, 
that repeated sterilization of the same soil may not be disadvantageous in 
certain cases and for other reasons not discussed here. 

Influence of temperature of soil following sterilization. The results obtained 
relative to the toxic or beneficial action of heated soils are found to be influ- 
enced to a considerable degree by the temperature of the moist soil following 
sterilization, and this may explain some of the variations which have been 
secured in different experiments carried on at different seasons of the year. 
Russell (64) and Koch and Luken (32) have previously noted that results 
secured on heated soils varied with the time of the year, although they did 
not apparently connect up this idea with soil temperature in particular. Pick- 
ering (50) in studying the loss of the toxic action of heated soils found that its 
temperature influenced the loss of toxicity to seed germination. It was noted 
by the writer, in connection with some experiments dealing with the influence 
of soil temperature on the development of the root-rot disease of tobacco 
(Thielavia basicola), in which steam sterilized soils were used as checks, that 
retarding action and chemical injury were considerably more marked at soil 



32 



JAMES JOHNSON 



temperatures below about 25°C. than at higher temperatures (plate 4, fig. 1). 
This toxic action was so great as to materially interfere with the progress of 
the experiments, and it finally became necessary to use special methods of 
sterilization to avoid it; although the toxic action could be materially reduced 
by proper methods of handhng the soil after heating and before planting. In 
order to study this action further experiments were conducted with virgin 
sandy loam soil heated to approximately 100°C. and planted to tomatoes in 
soils kept at various temperatures together with checks in unheated soils. The 
tomatoes in unheated soils at soil temperatures below 20°C. made a poor 
growth and showed increased pigmentation, while those at soil temperatures 
above 23°C. and up to 32°C. made a good growth (plate 4, fig. 2). In the 
heated soils the tomatoes at 23-24°C. were practically killed by the toxic 
agent. The toxic action at lower temperatures was also great but not as 

TABLE 20 

Effect of repeated heating of soil on successive days to 115°C. on yield of crop grown 







DRY WEIGHT 




SOIL TREATMENT 


Muck soil (wheat) 
average of 
duplicates 


Virgin sandy loam 

(wheat), average 

of duplicates 


Greenhouse com- 
post (tomatoes), 
average of 
triplicates 


Not heated 


gtn. 
4.75 

9.10 
7.70 
8.80 

6.55 


gm. 
4.85 

7.35 
6.90 
5.65 

7.00 


gm. 

3.97 


Heated once 


9.73 


Heated twice 


9.00 


Heated /our times 


7.50 


Heated five times 


8.17 


Heated eight times 









marked as at 23-24°C. At a temperature of 27-29°C. practically no "chemi- 
cal" injury occurred, although some retardation in growth took place. It is 
very evident from this and other experiments that the results secured with 
heated soils are to a great extent influenced by the temperature of the soil. 
This response is believed to be due largely to differences in bacterial activity 
as influencing loss of toxicity of the heated soil. The reasons for this 
explanation will be evidenced by the experiments described later. 

Other factors concerned. In addition to the factors already mentioned as 
being influential in determining growth on heated soils, several others would 
bear discussion at this time. One of the most important factors, which will 
be left for later discussion, is the reinfestation of the soil with normal soil 
flora which has a fundamental influence on the toxic and beneficial properties 
exhibited. Among the minor factors should be mentioned that of the mois- 
ture content of the soil both during heating and during subsequent plant 
growth. Excessive moisture in the soil during heating as well as during plant 
growth seemingly predisposes the plant to the injurious action, or increases 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 33 

the amount of the toxic property in the soil. At any rate watering should be 
carefully regulated in heated soils, especially in experimental work. This 
is particularly true for the reason that once a sterilized soil is overwatered, 
great difficulty may develop in attempts to dry it out rapidly. 

The period between the time of heating and the time of planting will influ- 
ence the amount of toxic and beneficial action exhibited, if the soils are kept 
under ordinary conditions for plant growth during that time, but not if the 
soils are kept dry. Whether atmospheric conditions except in so far as they 
influence soil environment are of any degree of importance in determining the 
extent of the action of heated soils is not yet known; although they presumably 
may show some influence as suggested by Russell and Petherbridge (64). 

Qualitative changes in plant growth on heated soils 

The differences to be found in the response of seeds and plants to heated 
soils, aside from the more common measurable phenomena, are frequently 
so marked as to distinguish the heated from the unheated soils. These differ- 
ences have been generally noted by practically all workers on sterilized soils 
in the increased vigor and deeper green color of plants grown on sterilized 
soils. Russell and Petherbridge (64) have gone considerably further in de- 
scribing these qualitative responses, noting especially the formation of purple 
pigments in plants grown on sterilized soils as well as changes in the character 
of the.root system. 

With respect to seed germination, aside from rate of germination, it miy 
first be noted that many seeds, especially seeds of leguminous plants, appar- 
ently become much more easily over-run with fungi when germinated on 
heated soils. This fact is probably due to the absorption of the favorable 
property for fungus growth from the heated soil solution. In soils heated to 
250°C., especially in those which are most toxic upon heating, the tip of the 
radicle may often become browned or blackened from contact with the surface 
of the heated soil. Lettuce seeds on the most toxic heated soils frequently 
germinate in an abnormal manner, in that the cotyledons emerge from the 
seed coat before the radicle. In other cases the radicle may make a long 
spindly growth, seemingly in an attempt to grow away from the toxic medium. 
The lack of formation of root hairs as compared with the abundant formation 
on unheated soils is especially noticeable at some stages of germination and 
growth on heated soils. If the seeds are allowed to continue germination on 
the surface of heated soils, it may often be noted that the roots instead of 
penetrating into the soil, grow along the surface of the soil. When roots are, 
however, forced to grow into certain sterilized soils in order to maintain the 
life of the plant, the roots may be much retarded in growth and may become 
short and stubby, without root hairs, frequently discolored as a whole or in 
local areas, or deeply split radially, partly decayed and sometimes entirely 
killed. This is especially true in sterilized soils grown to plants under sterile 

SOIL SCIENCE, VOL. VII, NO. 1 



34 JAMES JOHNSON 

conditions. It seems reasonable to suppose, therefore, that the retarded 
early growth of plants on heated soils may in some instances be due in part 
to the failure of the root system to penetrate the soil with the consequent 
reduction of their functions, rather than to the absorption of the toxic principles 
of the plant. This point is illustrated in plate 5, figure 1 showing the retarda- 
tion of the root system of tomatoes grown at different soil temperatures. 
That toxic principles are absorbed is shown by the cases of ''chemical" injury 
obtained. That absorption of toxic properties occurs is also shown in seed 
germination. Certain seeds, of which lettuce may be cited as an example, 
behave very peculiarly on the most toxic of the heated soils, heated soil ex- 
tracts, or products of dry distillation of soils as already referred to. This is 
shown especially by the swelling of the seed to two or three times its normal 
size, together with a decided change in color of the interior seed coat to a 
greenish black. The seed in such cases is practically always killed and 
sometimes bursts the outer seed coat due to internal pressure. The inner 
seed coat is very tense and hard, and upon pressure, bursts, exuding a droplet 
of clear liquid. This reaction with lettuce seed is sufficiently characteristic 
to make it valuable as a qualitative test of toxic agents and will be referred 
to as such in the consideration of the nature of toxic properties produced in 
heated soils. 

The qualitative responses of growing plants to heated soils are numerous 
and varied in type. Russell and Petherbridge (64) have made some obser- 
vations on this, especially with tomatoes. With regard to the formation of 
a purple pigment the impression is left that the pigmentation is characteristic 
on heated soils particularly. This does seem to be the case, however. Toma- 
toes grown on a wide variety of soils and under several different conditions may 
produce excessive purple pigmentation. Soils low in fertility, or cold soils es- 
pecially, seemingly respond in a similar manner to heated soils in pigment pro- 
duction. Purple pigmentation on heated soils is not pecuUar to tomatoes alone, 
but seems to be a response more or less common to other plants, being especi- 
ally noticeable in cabbage and lettuce, as far as observed in these experiments. 
Although pigmentation sometimes occurs on heated soils in case of tomatoes, 
it is not necessarily associated with heating. It may occur in the early plant 
growth, and finally disappear in the later growth; its persistence when pres- 
ent, apparently is in proportion to the unfavorableness of the soil for plant 
growth. 

Another type of color reaction which has been noted in the case of tomatoes, 
but which may be associated with stimulated as well as retarded growth, is a 
yellowish green mottling or mosaic appearance of the leaves resembling to 
some extent the Mosaic disease but usually without malformations (plate 6, 
fig. A). This abnormahty also usually disappears in a short time and is ap- 
parently associated with diminished activity of the chlorophyll bodies due 
to the absorption of a toxic substance fron the soil. In other plants such as 
tobacco, mottling may not occur but rather a uniform yellowing, especially 
at the leaf tips or margins. 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 35 

The most marked efiFect of heated soil on plant growth, however, is that 
which has been termed "chemical" injury. This is characterized by distinct 
spotting of the leaves or lesions on the stem, sometimes the latter occurring 
at the surface of the soil and resembling the damping-oflF disease. These 
spots or lesions often become confluent and the entire leaf or plant may wither 
and die. In some instances this action has been observed to become local- 
ized just below the terminal bud, resulting in the death of the latter and 
consequently the premature branching of the plant. In other instances 
"chemical" injury appears in the early stages of growth of the plant with 
the result that they may be killed outright or remain for weeks or months 
without making appreciable growth. Where the action has not been too 
severe, however, the plants may entirely recover and produce greater yield 
than in unheated soil. On the other hand this type of injury has been ob- 
served in plants which apparently made normal growth during the early 
stages of growth but finally showed symptoms of chemical injury in a la'te stage 
of growth (plate 5, fig. 2). The first signs in such cases are sometimes in the 
veins and midribs of the leaf, and are exhibited by the browning of the veins 
and curling of the leaves or at other times by spots which become confluent 
resulting in the entire leaf drying and dropping from the stalk. This con- 
dition has all the appearances of a disease caused by a parasitic organism. 
On account of the fact that it is not always associated with retardation, it 
appears that it is not necessarily the result of a product formed during heating 
of the soil but may be the result of a substance developed subsequent to 
heating. It is, however, also probable that the explanation of this response 
occurring several weeks after heating the soil may be due to the occurrence 
of certain environmental conditions favorable for its production or mani- 
festation, as, for instance, modified soil temperature or moisture conditions. 
This conclusion is supported by the previously mentioned soil temperature 
experiments and by the observation that overwatering heated soils may 
increase this type of toxic action. 

Similar "chemical" injury which may be mistaken for diseases of para- 
sitic origin have been found to occur upon several other plants grown on 
heated soils, particularly upon soybeans, cow peas, and tobacco. In the 
case of soybeans, the browning of the veins of the leaf is especially striking. 
If this action affects the leaves when they are young and in a rapidly growing 
condition, it results in leaf curling (plate 6, fig. E). 'The condition is similar 
to that sometimes occurring on tomatoes. The spotting of the leaves of soy- 
beans on heated soil usually occurs on the leaf margins and is seemingly as- 
sociated with the rapid transpiration at these points (plate 6, fig. D). In 
the case of cow peas, the spotting is far less distinct on the leaves, the spots 
being much smaller, usually not larger than a pin head, and raised rather 
than sunken (plate 6, fig. F). In the case of tobacco, the spots resemble very 
much certain leaf-spots occurring in the field, some of which are of bacterial 
origin and others probably of a non-parasitic nature (plate 6, fig. C). 



36 JAMES JOHNSON 

These qualitative responses are much more marked in some soils than in 
others. They may occur in a wide range of soil types and have seemingly 
no relation to the nitrogen content of the soil. They are influenced by 
environmental conditions of the soil especially, but they do not appear to be 
necessarily associated with degree of toxicity as represented by retardation 
of plant growth in heated soils, in view of the fact that they may appear on 
plants benefited in their growth by heated soils as well as on plants which 
are retarded. The relation of soil temperature to this toxic action has been 
especially demonstrated in the case of tomatoes and tobacco. It was found 
that "chemical" injury to tomatoes and tobacco on heated soils usually oc- 
curred at soil temperatures between 15° and 25°C. and not at higher or lower 
temperatures. In later experiments, however, it became evident that this 
injury might occur at lower or higher temperatures if other conditions, such 
as greatly increased moisture content of the soil, occurred. All observations 
point toward the fact that a fairly delicate relation exists between the extent 
of "chemical" injury and the balance between soil moisture and soil tempera- 
ture. In the acid sand (lime requirement 9.38 tons per acre) the "chemical" 
injury was marked following sterilization. If neutralized with calcium car- 
bonate before sterilization, this injurious action did not occur. All evidence 
has gone to show that this toxic action is most marked in acid soils following 
sterilization. 

Other minor qualitative responses of plants to heated soils occur, such 
as increased tendency toward branching in the case of soybeans. Under 
greenhouse conditions at least, the soybeans normally drop most of the lateral 
buds as well as the lower leaves and the plants tend to grow tall and spindly 
rather than short and bushy. Although this condition may be especially as- 
sociated with infertile soils, it is seemingly also associated with reduced light. 
In heated soils, however, these lateral buds do not fall off as readily in most 
instances but persist and produce short stout leafy branches deep green in 
color, even in cases of marked retardation due to toxic action. 

Some mention is to be found in the literature of increased susceptibility of 
plants to disease as a result of being grown on heated soil. Wilson (78), for 
instance, reports wheat rust {Puccinia graminis) and wheat mildew (Erysiphe 
graminis) more serious on weakened plants grown on soils heated to high 
temperatures, than on more vigorous plants grown on soil heated to lower 
temperatures. The probable changes in susceptibility to disease is to be 
considered in connection with a separate paper on the use of heated soils in 
phytopathological research. 

The development of fungi in heated soils 

That certain fungi grow very well on burned-over areas has probably been 
noted, with only passing comment, for a very long time. Tacke (74), however, 
in studying the effect of the heating of soil on the solubility of nitrogen, noted 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 37 

that fungi grew very well on the extracts of heated soils and believed that 
this was due to the excellent nitrogen supply present. Kurzwelly (39) re- 
ported that various species of fungi not living in soil before sterilization 
flourish vigorously afterwards. Kosaroff (35) noted, especially, the develop- 
ment of Pyronema confluens on heated soils where it did not occur on unheated 
soils. He concluded that the extraordinary development of this fungus on 
heated soil was not due to the resistance of the spores of the fungus to the 
sterilizing action but rather to the destruction of a substance toxic to the de- 
velopment of Pyronema in ordinary soils. Seaver and Clark (67, 68) began 
a series of investigations on the development of Pyronema omphiloides on 
heated soils, in an attempt to explain the reason for this behavior and incHned 
toward the belief that the beneficial action upon fungi was due to the increased 
concentration of the food supply. 

In the open and especially in the greenhouse in the presence of air-borne 
spores, the heated soils rapidly became covered with a thick mat of Pyronema 
confluens (Tul.) or Pyronema omphaloides (Bull.), Fuckel. Under favorable 
environmental conditions the soil may on the other hand become overrun 
with various ordinary molds, especially those of Penicillium and Mucor. 
It appears, however, that heated soils are considerably more favorable for 
Pyronema than for Penicillium and other fungi, but no more favorable for 
the latter than for other forms. The common occurrence of their spores in 
the air, probably explains the presence of certain fungi on the heated soils in 
many instances. Where other fungi, such as Rhizoctonia, Pythium, Thie- 
lavia, Fusarium, and other organisms commonly inhabiting soils are reinocu- 
lated into heated soils, they apparently find it a very much more favorable 
medium for growth than unheated soils. 

Although no detailed experimental work concerning the relation of fungi 
to heated soils was carried on in connection with the experiments of the writer, 
yet considerable observational data are at hand, which are seemingly in some 
instances contradictory to some of the conclusions of the above named authors. 
The relation of the following factors in regard to the growth of fungi upon 
heated soils has been especially noted: temperature to which soil is heated, 
influence of soil type, influence of soil reaction, length of time of heating, 
growth on soil extracts, and the effect of soil temperature and moisture on 
fungus growth in heated soils together with certain correlations between fun- 
gus growth and various other factors studied. The observations have shown 
that the growth of fungi in soils heated to different temperatures is best at 
about 250°C., the growth either occurring later or being less profuse or en- 
tirely absent in soils heated to higher or lower temperatures (plate 7, fig. 1). 
Tlie range for the development of Pyronema appears to be from about 100°C. 
to 350°C., temperatures above or below this usually giving no growth what- 
ever. This is seemingly true for practically all soils studied, but much more 
marked in some than in others. It should be noted at once in this connection 
that there is a close correlation between this growth and various other phe- 



38 JAMES JOHNSON 

nomena studied in soils heated to different temperatures, i.e., toxicity to seed 
germination, early plant growth, and beneficial action to late plant growth. 
It may be, therefore, that the property which is favorable for the develop- 
ment of Pyronema is the same which is toxic to seed germination and early 
plant growth as believed by Seaver and Clark (68), but no further evidence 
of this theory could be found; while on the other hand some contradictory 
evidence has been secured. 

The extent to which fungi, especially Pyronema, develops varies greatly 
for different soils when heated to 250°C., it being on the whole greater on those 
soils high in organic matter and less in those low in organic matter, although 
no definite proportion in this respect has been established. Comparing the 
development of Pyronema on the various soils used, it was usually found to 
be most profuse on the peat and least profuse on the Norfolk sand. It was 
interesting to note that Pyronema in one instance grew most profusely on peat 
soil heated to 250°C. which was so toxic to tobacco and other plants trans- 
planted into it, that they were killed in a few days. It has been observed, 
however, that Pyronema has made an appreciable growth upon red clay and 
upon practically pure sand heated to 110°C. The relatively low concentra- 
tion of the soil solution in these cases raised the question as to the importance 
of concentration of soil solutions in connection with fungus growth on these 
soils, and this point will be discussed briefly in a later paragraph. The re- 
action of the soil within wide ranges (lime requirement of 9.38 tons per acre 
to one with corresponding alkalinity) seemed to have no influence on the rate 
of growth of Pyronema. In regard to the influence of moisture supply on the 
development of Pyronema on heated soils, it was found that the best develop- 
ment was on soils kept at one-half saturation. Somewhat less growth oc- 
curred on soil kept at one-fourth saturation, followed by that at three-fourths 
saturation with relatively poor growth. No growth was obtained on soil 
kept at full saturation. No growth, of course, is obtained on soil which is 
air dry, but it should be especially noted that the property favorable to the 
development of Pyronema may be kept indefinitely in air dry soils, whereas 
it is relatively rapidly lost in moist or wet soils. 

The time of heating the soil has some influence on the growth of Pyronema, 
it being found for instance to occur first on soil heated to 115°C. for 160 min- 
utes, and then appearing gradually on soils heated for 80, 40, 20, and 10. min- 
utes. In this case no correlation seemed to exist with the growth of tomatoes 
on this soil. It has generally been found that an appreciable increase in con- 
centration of soil solution and ammonia occurs on continued heating of soil 
at 115°C. The development of Pyronema upon the surface of soils main- 
tained at different constant temperatures offers some interesting suggestions. 
At temperatures above 28°C. no growth of Pyronema has been observed. 
At temperatures ranging between 20° and 28°C., Pyronema usually appeared 
soon after the heated soil had been moistened, fruited, and then disappeared 
in a relatively short period of time. Finally growth appeared gradually upon 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 39 

the soil at lower temperatures, and at temperatures of 10° and 15°C., the fun- 
gus persisted and grew for several weeks, an occurrence which is relatively 
uncommon under ordinary conditions. The late appearance and slower 
growth at the lower temperatures is, no doubt, partly due to a temperature 
relation effecting the organism itself, but it is also reasonable to suppose that 
the persistence of Pyronema at these lower temperatures is due also to the 
maintenance of the favorable property in the heated soil, and its early dis- 
appearance at the higher temperatures is due to the rapid loss of this favorable 
property for its growth. 

That heated soils are also favorable to the growth of bacterial organisms 
is evidenced by the fact that extracts of heated soils left exposed to the air 
or inoculated with bacteria soon become teeming with organisms. Lodge 
and Smith (41) in experiments with decoctions of sterilized and unsterilized 
soil found that bacteria may or may not be favored in growth by the sterilized 
soil decoction, depending upon the nature of the soil. The extract of soil 
heated to 250°C. has been repeatedly noted in my experiments to be more 
favorable to the growth of bacterial organisms than extracts of the same soil 
heated to higher or lower temperatures. This has also been shown by making 
up soil extracts with 2 per cent agar and inoculating tubed slants with pure 
cultures of Bacillus suhtilis. The growth in heated soil extract agars was best 
in the 250°C. extract and poorer in the higher or lower temperature extracts. 
The growth of Penicillium in sterilized soil extracts from heated soils was ap- 
proximately in the same proportion and in the following order: 250°C. extract 
best, 200°, 150°, 100°, 350°, 50°, and unheated poorest. As noted by Seaver 
and Clark, it is practically impossible to keep soil extracts from being very 
strongly invaded with lower microorganisms without keeping them under 
sterile conditions. 

The nature of the toxic and beneficial substances produced on heating 

The earliest theories in regard to the cause of the injurious action of heated 
soils were largely founded on the effect of heat on the physical properties of 
the soil, particularly on the destruction of vegetable matter in burning, al- 
though Davy (12) as early as 1819 did not agree with these theories. Arends 
(1) believed that bad results follow burning due to exhaustion of fertility. 
Struckmann (73) suggested that certain inorganic substances may be produced 
which are injurious. Dietrich (13) was first to carefully observe the injurious 
action of heated soils, and concluded that there was a poison formed from the 
alteration of the organic matter, and that according to the amount of this 
poison formed and the sensitiveness of the plant to it, either the injurious or 
the beneficial action predominated. Where the plants were less susceptible 
to the poison, there was observed an increased absorption of soluble nitrogen 
by the plants. 

Schulze (66) found a harmful effect of steam sterilization of soil which he 
attributed to increased solubility of the humus. The effect was found to vary 



40 JAMES JOHNSON 

with different soils and plants, and to be more marked in early stages of plant 
growth. 

Pickering (50) in 1908 published the first of a series of four papers on the action 
of heated soil on seed germination and plant growth and the changes occurring 
in the heated soils. His work was especially concerned with the toxic action 
of the heated soil, although no special attempt was made to determine 
any definite injurious compound. Extensive studies were made particularly 
concerning the relation of the increased solubihty of organic and inorganic 
material in the soil due to heating. These papers are of sufficient importance 
in connection with the investigations reported here to briefly review each 
separately. Pickering's conclusions were more or less modified in each suc- 
ceeding paper, however, and it is somewhat difficult to determine to which 
points most significance should be attached in a brief review. 

Pickering's first paper (50) brought out some very interesting conclusions 
which are further extended in the following papers. The unfavorable effect 
on seed germination was found to be proportional to the content of organic 
matter when different heated soils were compared. In general the "richer" and 
more favorable the soil for germination before heating, the more inhibitory 
it becomes after heating. He beUeved that the destruction of the inhibitory 
substance in heated soil, as a result of added extracts of unheated soil, is due 
to a process of oxidation. No appreciable destruction of the toxic property 
resulted when soil was kept for several months at a low temperature in a mod- 
erately dry condition. At higher temperatures, however, in presence of mois- 
ture, some of the inhibitory properties were lost, probably through oxidation. 
The inhibitory substance was found not to be of an acid reaction. The retard- 
ing effect could not be explained by alteration of bacterial flora in the soil 
as the alteration extends progressively beyond that necessary to destroy 
all bacteria. The maximum of toxic effects to seed germination was found 
to be produced on heating to 200°C., although in a second paper (51) 250°C. 
was said to be the optimum temperature for toxicity. In this paper Picker- 
ing also concludes, contrary to his first paper, that the "poverty" or "rich- 
ness" of a soil under ordinary conditions bears no relation to its behavior 
when heated. There appeared to be no connection between the total nitrogen 
in the soil and the extent to which it was altered by heat, and the inhibitory 
substance produced was not apparently the same in all cases. Pickering's 
third paper (52) concludes, relative to the nature of the toxic agent, that 
the increase in soluble matter produced by heating a soil and the accom- 
panying toxic properties detrimental to the germination of seeds are gradually 
reduced by exposing the soils in a moist condition to the air, even under asep- 
tic conditions, but are not reduced when the soils are kept moist in the absence 
of air. The destruction of the toxic substance is therefore thought to be due 
to oxidation. 

In a fourth paper (53) concerning plant growth in heated soils, Pickering 
concludes that the substances favorable for plant growth and the substances 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 41 

unfavorable for plant growth in heated soils are of a different nature. The 
toxic action on plant growth is beUeved to disappear under the influence of 
oxidation as it does in the case of seed germination as noted above. Whether 
or not the toxic agent to seed germination and plant growth is identical can- 
not be settled but it is provisionally assumed that they are the same. As a 
result of growing difiFerent kinds of plants in heated soils, Pickering concludes 
grasses are more resistant to the toxic action than non-grasses. 

Schreiner and Lathrop (65) heated soils to 135°C. under steam pressure and 
found an increase in various organic constituents not ordinarily considered in 
questions of soil fertiUty. Some of these were beneficial to plant growth and 
one in particular, dihydroxy stearic acid, was found to be harmful to pliant 
growth. These authors are inclined to explain the injurious action produced 
on sterilized or heated soils to the formation of this toxin and the beneficial 
action to the formation of other organic compounds. Although the writer 
attempted to isolate dihydroxystearic acid from the most toxic of the 
heated soils studied without success, the main objection to this theory in con- 
nection with explaining the response of plants to highly heated soils seems to 
be that it is highly probable that this compound having a low dissociation 
temperature, would be practically destroyed before a temperature of 250°C. 
is reached. 

The objection to Fletcher's theory (18) that heated soils do not produce 
toxins, which delay the germination of seeds, but that this delay is caused by 
decreased imbibition of water from strong solutions of heated soils seems to 
be contradicted by the observations made in connection with the experi- 
ments described in this paper where it was found that certain seeds actually 
imbibed more water than in normal germination. 

Russell and Petherbridge (64) present a considerable amount of data bear- 
ing upon the retardation and stimulation of seed germination in soils heated 
to low temperatures and treated with antiseptics. They made no study, how- 
ever, of this problem in particular, but were concerned mainly with the bene- 
ficial action of sterilized soils upon plant growth. These authors disagree with 
Pickering as to the chemical nature of the toxic substance and state that they 
could not find proof that the harmful effect of heated soils on germinating 
seeds passed off after a time. That ammonia might be the toxic agent pro- 
duced was suggested, but they could find no analytical data to show that any 
relation existed between it and the amount of retardation or acceleration to 
seed germination. 

Lyon and BizzeU (42) have also made a careful study of the substances pro- 
duced in steamed soils injurious to plant growth. They found that the time 
required for the various soils to recover from this injurious action was, with 
one exception, in the order of their relative productiveness. Steaming was 
found to increase the soluble nitrogenous compounds and also phosphoric 
acid. The nitrates were reduced to nitrites and ammonia, but most of the 
ammonia was formed from organic matter. They concluded that the toxic 



42 JAMES JOHNSON 

substance produced is a controlling factor in productivity of steamed soils. 
Apparently, however, they do not suggest anything further as to the chemi- 
cal nature of the injurious property. 

Practically all the results of previous investigations on the nature of the 
toxic agent in heated soils have been based on results secured on heating be- 
low the "critical" temperature. It seemed to the writer, therefore, that a 
detailed study of soil heated to the optimum for the production of the injuri- 
ous action might lead to conclusions more satisfactory than those secured 
from lower temperatures of heating. That the substance toxic to seed germi- 
nation increases with the rise in temperature up to about 250°C., and 
thg,t on further heating, the toxic property is gradually lessened until it has 
entirely disappeared before a temperature of 500°C. is reached, has repeatedly 
been found to be true. It is reasonable to assume, at least, that at about 
250°C. a balance is reached between the maximum production and maximum 
retention of the toxic substance by the heated soil. Whether the substance 
is destroyed at temperatures above 250°C. or merely volatilized is not exactly 
clear. There is some reason to presume, however, that the toxic substance 
is gradually produced in increasing amounts up to 250°C. and beyond, but 
that it is rapidly volatilized above 250°C. 

Wafer extracts of heated soils 

Water extractions were made of soils heated to various temperatures and 
the comparative rate of seed germination obtained. The results for water 
extracts of Waukesha silt loam are shown in table 21, where it may be seen 
that the toxic property is at least partly soluble in water. Considerable vari- 
ation is shown in the germination percentages after 24 and 42 hours. This 
may be due in part to experimental error, but is also seemingly a further 
indication of similar conditions repeatedly noted in which certain soil 
heated to 50°C. are more injurious to germination than unheated soil or soils 
heated to 100°C. This condition has been found to recur too often to be 
considered merely accidental and yet no explanation can be ofifered for it at 
this time as it appears to bear no consistent relation to any of the changes in 
the soil which have been studied. The germination after 66 hours, how- 
ever, as shown in table 21 does suggest that the amount of toxin soluble in 
water is in proportion to that in the soil itself. In some cases the extraction 
of a soil heated from 200°C. to 300°C. with an equal weight of water yields 
an extract considerably more toxic than the soil itself. Such marked toxic- 
ity is not usually obtained, however, but it is not improbable that the in- 
creased injurious action of the extract, beyond that of the soil itself, is due to 
the separation of the toxin from the ameliorating effect of the absorptive power 
of the soil. 

In order to arrive at some approximation of the proportion of the toxic mat- 
ter extracted with water in repeated extractions of the same soil, the experi- 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



43 



ment detailed in table 22 was performed, two different soils being used. The 
results show that a large proportion of the toxicity was removed at the first 
extraction and that the second and third extractions with water yielded con- 
siderably less of the toxic property. Since the property toxic to seed germi- 



TABLE 21 



Relative rate of germinatioti of seed on water extracts of soil heated to different temperatures; 
Waukesha silt loam; lettuce seed 



TREATMENT 


GERMINATION AFTER 


TOTAL 




24 hours 


42 hours 


66 hours 


90 hours 


1 14 hours 




Not heated 


per cent 
30 

3 
24 

1 




34 

2 


per cent 
93 
68 
90 

7 
13 

1 
93 
41 


Per cent 

99 

98 

99 

74 

50 

17 

99 
100 


per cent 

99 
100 

98 

79 

76 
100 


per cent 

99 
92 
97 


per cent 
99 


Heated to 50°C 


99 


Heated to 100°C 


100 


Heated to 150°C 


99 


Heated to 200°C 


92 


Heated to 250°C 


97 


Heated to 300°C 


100 


Heated to 350°C 


100 







TABLE 22 

The toxicity of repeated water extractions of soil heated to 250°C. on seed germination; lettuce 

seed 



Waukesha silt loam . . . 



Virgin sandy loam . 



TREATMENT 



Check 



Heated 



Check 



Heated 



EXTRACTION 




GERMINATION AFTER 




42 
hours 


66 
hours 


90 
hours 


114 
hours 


138 
hours 




per cent 


per cent 


per cent 


per cent 


Per cent 


First 


77 


96 


97 


98 




First 


1 


2 


17 


76 


78 


Second 


11 


40 


59 


74 




Third 


57 


80 


85 


92 


93 


First 


98 


99 








First 


2 


7 


26 


69 


76 


Second 


9 


50 


65 


87 


89 


Third 


45 


82 


86 


93 


94 



per cent 

98 



79 

75 
94 

100 

77 
89 
94 



nation is readily soluble in water, further study upon heated soil extracts was 
made. 

That this toxic principle is extractable from all types of heated soils is 
shown in table 23, although it is not proportional to the toxicity exhibited by 
the soils themselves. Peat and muck and especially the fine sandy loam, 
for instance, ordinarily do not exhibit as great a toxicity to seed germination 
as Waukesha silt loam. The extracts of these first-named soils, after hav- 



44 



JAMES JOHNSON 



ing been heated, are, however, considerably more toxic than the heated soils 
themselves, and slightly more toxic than the extract of Waukesha silt loam. 
This is especially significant when it is remembered that the concentration of 
the soil solution used is far less than that actually existing in the soils them- 
selves. The absorptive capacity of the soil, then, has seemingly a most pro- 
found influence not only upon the total toxicity exhibited by a soil but also 
upon its relative toxicity, and this is apparently roughly correlated with the 
total amount of organic matter in the soil. 

TABLE 23 

Relative rate of germination of seed in water extracts of different soils heated to 250°C.; lettuce 

seed 



Distilled water .... 

Peat 

Muck 

Waukesha silt loam 
Fine sandy loam . . . 
Virgin sandy loam . 

Clay 

Norfolk sand 







GERMINATION AFTER 




42 
hours 


66 
hours 


90 

hours 


114 
hours 


138 
hours 


152 
hours 


176 
hours 


per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


60 


78 


82 


87 


90 





















2 


6 








2 


5 


20 


27 


35 





4 


20 


44 


65 


72 


76 


1 


3- 


9 


26 


53 


63 


69 


2 


8 


32 


53 


76 


80 


82 


2 


10 


19 


33 


61 


70 


73 


21 


51 


73 


87 


89 


91 


93 



200 
hours 

per cent 

90 

9 

39 

81 
75 
82 
78 



TABLE 24 

Seed germination in distillates from extract of heated soil. Waukesha silt loam heated to 250°C.; 

lettuce seed 





GERMINATION AFTER 






24 
hours 


48 
hours 


72 
hours 


96 
hours 


120 
hours 


144 
hours 




Check (distilled water) 


per cent 

89 

2 
1 
4 



per cent 

99 

4 

3 
11 

5 


per cent 

14 
13 
38 
31 


Per cent 

36 

22 
63 
80 


per cent 

44 
48 
84 
89 


per cent 

45 
63 
90 
92 


per cent 


First (gaseous) 


45+ 


Second 


66 


Third 


92 


Non-volatilized 


92 



Further evidence upon the nature of the toxic property is obtained by dis- 
tilling the soil extract. A rough fractionation was made of a water extract 
of Waukesha silt loam heated to 250°C. The fraction (table 24) which came 
over before the soil solution came to the boiling point showed almost as much 
toxicity to seed germination as the second portion and considerably more than 
the third, which came over after boiling. That all the toxic substance was 
not volatile is shown by an active toxic principle apparently still remain- 
ing in the distillation flask. 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 45 

Further evidence of the volatility of the toxic principle in heated soil was 
procured in several different ways. Heating soil in closed vessels usually pro- 
duced more properties toxic to seed germination than heating in open vessels. 
Drawing a current of air through the soil into water while heating yielded 
toxic properties in the water. Drawing air through the dry soil after heating 
also removed some of the toxic principle. 

It has been hoped that the difference in reaction of seeds of different species 
of plants would give some clue to the nature of the injurious action. Several 
experiments were therefore planned for the purpose of comparing the response 
of different seeds to heated soils and to known chemical substances. 

The difference in response of seeds of various kinds to the toxic action of 
heated soil has already been referred to. In an effort to determine if this re- 
action bore any relation to the genetical relationship of the various groups of 
plants, a germination test was performed using several different species of 
the same family in comparison with various species of other families (tables 
25 and 26). The results indicate that a considerable degree of correlation ex- 
ists in this regard. The Gramineae are characteristically "resistant" to the 
toxic action as are also the Cticurhitaceae. The Solanaceae and Legumino- 
seae are, however, characteristically quite " suscep'tible " to the injurious ac- 
tion, although not so much so as lettuce seed. The data at hand are not suf- 
ficient to satisfactorily estabhsh this rule even for the families mentioned and 
cannot, of course, be said to apply to other families of plants although there 
is a suggestion and natural expectation that this may be true. It is- interest- 
ing to note here that oats and rye showed some degree of stimulation on 
Waukesha silt loam heated to 250°C., an occurrence which illustrates the 
striking resistance of these species to the injurious action as compared with 
lettuce or clover. On the assumption that different seeds respond differently 
to different chemicals, it seemed reasonable, therefore, that if a considerable 
degree of correlation could be found between the germination of different 
seeds on heated soil and their germination in the presence of certain pure 
chemical substances of known strength, it would be possible to arrive at some 
conclusion regarding the composition of the injurious product produced on 
heated soil. 

Production of ammonia in heated soils 

Of the substances commonly present in soils, which are at the same time 
toxic in relatively very .small amounts and which have further more been 
frequently found to be toxic to plants in soil (14), ammonia is probably the 
most common. Various investigators have shown a small initial production 
of ammonia in soil on sterilization. Herbert (24) as early as 1889 noted am- 
monia production in soil on heating to 100°C., while Kelley and McGeorge 
(30) and the results reported in this paper as well as that of others have 
shown large increases on heating to higher temperatures. It was logical, there- 
fore, to closely examine the relation of ammonia to the toxic action of the 



46 



JAMES JOHNSON 
TABLE 25 



Relative rate of germination of seeds of different families of plants on heated soil; Waukesha 

silt loam soil heated to 250°C. 





SOIL 


GERMINATION AFTER 




FAMILY AND SEED 


TREATMENT 


45 
hours 


79 
hours 


90 
hours 


114 
hours 


138 
hours 


162 
hours 








per cent 


per (ent 


per cent 


per cent 


per cent 


per cent 


per cent 


Gramineae 


















/ 


None 


76 


85 


87 








87 


Rye 1 


Heated 


81 


85 


86 








86 


Wheat 1 


None 
Heated 


57 
31 


96 
91 


98 
98 








98 
98 


Barley i 


None 
Heated 


70 
51 


97 
95 


99 
98 








99 
98 


Oats I 


None 
Heated 


8 
10 


75 
88 


97 
98 








98 
99 


Corn i 


None 


8 


28 


55 


89 


94 


96 


97 


Heated 


3 


15 


46 


74 


89 


94 


98 


Cucurbitaceae 


















Cucumber < 


None 
Heated 


35 
21 


82 
74 


87 
85 


88 
88 






89 


Squash < 


None 
Heated 


36 
4 


81 
38 


91 
63 


71 


72 


73 


91 

81 


Pumpkin < 


None 
Heated 


66 
11 


87 
51 


96 

82 


98 
93 


94 


95 


98 
95 


Muskmelon < 


None 
Heated 


25 
4 


51 
13 


72 
34 


73 
40 


76 
50 


77 
66 


77 
69 


Watermelon I 


None 
Heated 






7 
2 


32 
17 


49 
31 


69 
56 


82 
85 


Sokmaceae 


















Nicotiana rustica < 


None 
Heated 




81 
17 


85 
60 


86 

72 


77 


81 


86 

84 


Egg olant { 


None 
Heated 




16 



38 



59 
1 


66 

8 


69 
36 


71 




72 


Pepper < 


None 




1 


3 


6 


12 


34 


51 


Heated 













3 


16 


55 


Tomato < 


None 




3 


32 


50 


57 


63 


69 


Heated 










7 


34 


49 


64 


Datura < 


None 
Heated 






21 



56 



69 

14 


75 
42 


76 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



47 



TABLE 26 

Relative rate of germination of seeds of the legume family on heated sail; Waukesha silt loam 

heated to 250°C. 



Red clover. 



Alfalfa. 



Bur clover. 



Tapary bean . . . 
Crimson clover . 



Goats rue. 



Robinia. 



Hairy vetch. . 
Geuge clover . 



Birdsfoot clover. 



Astragalus . 



Peas. 









GEEiriNATION AFTER 








SOIL 


















TREATMENT 


45 
hours 


66 
hours 


90 

hours 


114 
hours 


138 
hours 


162 
hours 


210 
hours 






per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


None 


41 


80 


86 










86 


Heated 





2 


4 


7 


11 


14 


21 


53 


None 


22 


43 


51 


52 








52 


Heated 


1 


6 


11 


15 


20 


25 


34 


41 


None 


21 


27 


31 


32 








32 


Heated 


8 


12 


13 


15 


15 


16 




18 


None 


13 


77 


84 


89 


91 






91 


Heated 





52 


80 


89 


91 






91 


None 




10 


14 


15 








15* 


Heated 










1 








1* 


None 




6 


10 


12 


13 






13* 


Heated 







3 


6 


10 


11 


12 


12* 


None 




3 


6 


9 


17 


20 


25 


25 


Heated 







2 


5 


14 


19 


23 


25 


None 




1 


9 


13 


15 


16 


17 


17* 


Heated 




1 


3 


12 


16 


17 




18* 


None 




2 


11 


21 


27 


29 


31 


31* 


Heated 













1 


2 


3 


3* 


None 






8 


10 


11 


12 




12* 


Heated 









2 








2* 


None 






8 


19 


30 


34 


40 


46* 


Heated 












1 


1 


2 


5* 


None 








20 


38 


77 


82 


82 


Heated 








6 


21 


58 


74 


74 



* Seeds molded and failure to germinate may be partly due to this, but the seeds were also 
low in germinating capacity. 

heated soil. That this increase is appreciable on heating to 115°C. and very 
striking on heating to 250°C. is shown by four separate determinations for two 
different soils in table 27. 

Two soils were now taken and heated to temperatures ranging from 50° to 
350°C. and the amount of ammonia produced determined. Here it was found 



48 



JAMES JOHNSON 



that on the whole there was a gradual increase in ammonia on heating to tem- 
peratures up to 250°C. On heating to higher temperatures, the ammonia- 
content was reduced (table 28). The close correlation between the ammonia 
content on heating to different temperatures, and the toxicity to seed germina- 
tion is evident from the data presented. The reduced ammonia content on 
heating to 60°C. has some semblance of an error in determinations, but on the 
other hand its frequent occurrence in the experiments together with certain 

TABLE 27 
Increase of ammonia in soils heated to 115°C. and 250°C. 







NITROGEN AS AMMONIA IN 100 GM. SOIL 


AVER- 


SOIL 


TREATMENT 




AGE IN- 




Exp. 1 


Exp. 2 


Exp. 3 


Exp. 4 


Average 


CREASE 






mgm. 


mgm. 


mgm. 


mgm. 


mgm. 


percent 


■ 


Not heated 


4.20 


6.93 


3.36 


6.58 


5.28 




Muck \ 


Heated 115°C. 


6.72 


8.32 


8.26 




7.77 


47 




Heated 250°C. 


31.15 


31.01 


25.06 


30.10 


29.33 


455 


r 


Not heated 


1.96 


1.68 


1.82 


2.9 


2.09 




Waukesha silt loam < 


Heated 115°C. 


3.50 


3.78 


4.34 




3.87 


85 


^ 


Heated 250°C. 


18.48 


13.72 


15.40 


17.0 


16.15 


672 



TABLE 28 
Increase of ammonia in soils heated to different temperatures 





NITROGEN AS AMMONIA IN 100 GM. SOIL 


SOIL TREATMENT 


Muck 


Waukesha silt loam 




Single de- 
termination 


Increase 


Average of 
duplicates 


Increase 


Not heated 


mgm. 

3.36 
1.54 
8.26 
14.42 
25.06 
5.88 
3.92 


per cent 

-118 

149 

329 

646 

66 

16 


mgm. 

1.75 
1.40 
4.06 
9.52 
14.56 
7.32 
• 3.36 


per cent 


Heated to 60°C 


-25 


Heated to 115°C 


132 


Heated to 200°C 


444 


Heated to 250°C 


732 


Heated to 300°C 


318 


Heated to 350°C 


92 







irregularities of seed germination and plant growth on some soils, as already 
referred to, leads to the conclusion that some correlation may exist even here. 
This may be illustrated further by results secured on heating soil for various 
lengths of time (table 29) where not only the ammonia content was lowered 
on heating 10 and 20 minutes, but also the concentration of the soil solution 
compared with longer periods of heating, and these changes were accompanied 
by reduced germination on the soil but increased germination in the soil 
extracts. 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



49 



On comparing the amount of ammonia produced in muck and Waukesha 
silt loam soil on heating them to dififerent temperatures (table 28), it is found 
that the ammonia content is uniformly higher in the muck soil than in the 
Waukesha silt loam. Muck soil is, however, never as toxic to seed germina- 
tion as Waukesha silt loam when heated to the same temperature. That 
this increase in ammonia occurs in all soils used and is at its maximum at 
about 250°C. has been frequently shown in these experiments. The increase 
on heating to 250°C. has usually ranged from 100 to 1,000 per cent for the dif- 
ferent soils. No results such as reported by Potter and Snyder (55) i.e., re- 
duced ammonia content on heating peat to 200°C., have been obtained. No 
great uniformity in results has been secured in toxicity produced on heating 
the same soil to a certain temperature at different times owing to the varia- 
tion in the actual amount of heat apphed which variation seems unavoidable 
where automatically regulated ovens can not be used in all cases. The rela- 

TABLE 29 

Effect of heating soil at 115°C. for different lengths of time; Waukesha silt loam; lettuce seed 









GERMINATION 


TIME OF HEATING 


NITROGEN AS 

NH3IN 100 GM. 

SOIL 


LOWERING 

OF FREEZING 

POINT 


On soil after 


On soil extract after 




42 hours 


66 hours 


90 hours 


24 hours 


48 hours 


72 hours 


minutes 


mgm. 


°C. 


per cent 


per cent 


Per cent 


per cent 


per cent 


per cent 


None 


3.0 


0.007 


97 


99 


100 





83 


99 


10 


2.1 


0.004 


54 


98 


100 


4 


84 


100 


20 


2.8 


0.001 


49 


93 


100 


11 


83 


97 


40 


3.5 


0.015 


67 


98 


99 


2 


59 


100 


80 


4.1 


0.018 


69 


97 


98 


3 


71 


95 


160 


4.6 


0.026 


62 


93 


97 


12 


78 


94 


Distilled water 












48 


96 


98 



tive results where dififerent soils were heated at the same time in the auto- 
claves or ovens, have, on the other hand, been remarkably uniform. It should 
now be stated that the ammonia content of dififerent soils heated to the same 
temperature seemingly bears no relation to their toxicity to seed germination. 
This conclusion is illustrated in table 30 showing the results secured on heat- 
ing dififerent soils to 115°C., although it might be shown better by soils 
heated to 250°C. The results are somewhat confusing because of the fact 
that different soils behave dififerently towards seed germination in their nat- 
ural or unheated condition. This is especially marked in the case of Sparta 
sand, and may be due to a toxic agent which is normally present in this soil, 
and which is more or less common in all soils, since seed germination is ap- 
parently always somewhat retarded on unheated soils or their extracts as com- 
pared to germination in pure water on filter paper. In any case, heating of 
the soil to certain temperatures appears usually to reduce the natural retard- 
ing influence to seed germination. 



BOIL SCIENCE, VOL. VU, NO. 1 



50 



JAMES JOHNSON 



If heated and unheated soils are extracted with water and the ammonia de- 
termined in the extracts,, it will be found that a relatively high amount of am- 
monia exists in the heated soil extract as compared with the unheated soil 
extracts (table 31). This suggests that the absorptive capacity of the soil 
for ammonia has been reduced by the heating. With weak hydrochloric acid 
more ammonia can be extracted, but this extract containing more ammonia 
is less toxic than the water extract. The toxic substance is then apparently 



TABLE 30 



Efed of heating different soils to 115°C. in autoclave on ammonia content and on rate of 

seed germination; lettuce seed 



SOIL 


TREATMENT 


NITRO- 
GEN AS 
NH3 IN 
100 GM. 
SOIL 


IN- 
CREASE 
IN AM- 
MONIA 


GERMINATION 


AFTER 


TOTAL 




24 hours 


42 hours 


66 hours 








mgm. 


per cent 


per cent 


per cent 


per cent 


per cent 




Check 


11.7 




43 


99 




99 


Peat S 


Heated 


15.0 


28.2 





48 


70 


75 


Muck \ 


Check 


4.2 




68 


99 




99 


Heated 


7.4 


76.2 





88 


98 


98 


Waukesha silt loam \ 


Check 
Heated 


3.8 
4.8 


26.3 


3 



87 
80 


95 
96 


96 




98 


Virgin sandy loam i 


Check 
Heated 


3.0 
4.1 


36.6 


45 
5 


100 
98 


99 


100 




99 


Fine sandy loam \ 


Check 
Heated 


2.4 
3.4 


41.6 


56 
2 


98 
93 


98 


98 


I 


98 


Sparta sand \ 


Check 
Heated 


1.9 
3.2 


68.4 




2 


54 
72 


80 
94 


91 
96 


Norfolk sand , i 


Check 
Heated 


1.6 
2.2 


37.5 


16 



88 
49 


91 

83 


92 




95 


Red clay < 


Check 
Heated 


0.5 
0.5 


0.0 


7 
1 


89 
94 


97 
100 


97 


I 


100 



neutralized by the acid, or in some manner rendered less injurious. This 
experiment suggests at once both the possible relation of free ammonia to the 
toxicity of heated soils and the relation of the absorptive capacity of the soil 
for ammonia to the injurious action. 

It is fairly clear, nevertheless, from the data presented thus far that am- 
monia determinations of the soil as such cannot lead to any clear conception 
of the relation of ammonia to seed germination. This is largely because of 
the fact that the toxicity of a chemical substance in a soil is in proportion 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



51 



to the absorptive capacity of that soil for the substance in question. The 
great absorptive power of soil for ammonia is well known. It is, in fact, 
recognized in farm practice that the use of ammoniacal fertilizers on soils low 
in absorptive capacity may be productive of toxic action on plant growth (14). 
Due consideration must be given this matter, therefore, in relation to heated 
soils. Several angles of the problem present themselves for consideration. 
Soils of widely differing absorptive capacities have been used in the experi- 
ments. The absorptive capacity of each soil is influenced to a considerable 
extent by the degree of heat applied. That this is not a simple relation is 
indicated by the marked alteration of most soils as regards absorptive capacity 
for water when heated (by moist heat especially) to temperatures of about 



TABLE 31 



Ammonia in extracts of heated soil and seed germination in distillates from extracts with mag- 
nesium oxide; heating to 250°C. and extracted with distilled water and 
2 per cent HCl;* lettuce seed 



Waukesha 
loam. . . 



silt 



Virgin 
loam. 



sandy- 



Distilled water 



TREATMENT 



Check, water extract 
Heated, water extract 
Heated, acid extract 

Check, water extract 
Heated, water extract 
Heated, acid extract 



NITROGEN 

ASNH3 

IN EXTRACT 

OF 100 

GM. SOIL 




GERMINATION 


AFTER 




24 
hours 


42 
hours 


66 
hours 


90 
hours 


114 
hours 


mgm. 


per 
cent 


per 
cent 


per 
cent 


per 
cent 


per 
cent 


0.2 


11 


88 


97 


98 




3.2 





17 


56 


60 


62 


5.1 





38 


74 


77 


81 


0.35 


39 


97 


98 


99 


100 


3.7 





7 


16 


21 


28 


5.5 



62 


16 
99 


25 


27 


41 



per 
cent 

98 
66 

87 

100 
38 
50 

100 



* The ammonia was determined from a composite of three washings of the soil in equal 
weights of solvent after standing 24 hours with frequent shaking in each case. The lettuce 
seed was germinated in a part of about 25 cc. of distillate collected in 5 cc. of water. 



80 to 115°C., compared with the very rapid absorption of water in soils heated 
to high temperatures. This may not be a problem of simple absorption, how- 
ever, but rather one of capillarity. In general the heating of soil reduces its 
capacity of absorption, although considerable disagreement may be found in 
literature in regard to this, due most likely to differences noted in capillarity. 
If decreased absorption is the rule then in addition to the variation in different 
soils, it is reasonable to expect that the toxic property produced in a heated 
soil will vary in one direction in proportion to its natural absorptive capacity, 
and in the other, in proportion to the degree of heating. The method of 
solving this problem, however, does not seemingly lie in attempting to obtain 
correlation between the absorptive capacities of the various soils under dif- 
ferent conditions, but in studying the correlation in respect to seed germina- 



52 JAMES JOHNSON 

tion where definite and varying amounts of a toxic agent are added to the 
soil itself; the main difficulty encountered in this procedure being, however, 
that it offers no satisfactory direct solution of the problem as between heated 
and unheated soils. 

Two preliminary experiments were made to arrive at a rough approxima- 
tion of the absorptive capacity of different soils for ammonia, as measured by 
its toxicity to seed germination, and the response of different seeds to different 
amounts of ammonia in soils. It will be seen from table 32 that the smallest 
amounts of ammonia added had no appreciable effect on either the muck or 
silt loam soil, but that higher amounts stimulated germination and that the 
highest application retarded germination considerably in Waukesha silt loam. 
Quartz sand with very low absorptive capacity showed retardation at the 
lowest percentage of ammonia used. In the same way, where different seeds 
were used on one soil (table 33), certain seeds showed slight stimulation where 
the smallest amount of ammonia used was present, whereas others showed 
retardation. Rye and buckwheat were more resistant to the injurious 
action of ammonia than clover or lettuce seed. Table 34 illustrates the 
same facts with a greater variety of seeds. Rye, seemingly the most resist- 
ant, was followed in order b)'^ wheat, buckwheat, and flax. Garden-cress 
was the most susceptible (lettuce was not used in this experiment), followed 
by clover, cabbage, and tomatoes. Beans, cucumbers, tomatoes, and Datura 
showed an intermediate degree of resistance. If now, the germination of 
these seeds on Waukesha silt loam heated to 250°C. is compared with the 
germination on soil to which ammonium hydroxide has been added, considera- 
ble similarity will be noted (table 35). With the strength of ammonia used 
in this experiment, we find that in the case of all seeds, the soils treated with 
ammonia were more toxic than the heated soils. However, in the case of flax 
which is very resistant to the toxic action of heated soil, the seeds were prac- 
tically all killed by the soil to which ammonia had been added. Wheat, 
buckwheat, and cucumber, which are quite resistant to the toxicity of heated 
soil, respond proportionally in the same manner to ammonia, and lettuce, 
garden-cress, clover, and cabbage, which are quite susceptible, respond in a 
similar way to ammonia. The difference may be noted that in the case of 
those seeds susceptible to ammonia, practically all the seeds were killed, 
whereas in the heated soil, the large majority gradually germinated. 

The seeming failure of flax seed in this experiment to fall in line with expec- 
tations led to a more detailed experiment to determine if this seed would 
correlate itself in some measure with the other resistant seeds with respect to 
germination on soil to which ammonia had been added. Garden-cress was 
chosen as a check seed, as it had shown itself susceptible to ammonia in the 
previous experiment, and was at the same time quite susceptible to the tox- 
icity of heated soil. Different amounts of ammonia were added to soil both 
as ammonium hydroxide and as ammonium carbonate. The amount of 
ammonia in these and in the heated and unheated samples was determined 



TABLE 32 
Action of different amounts of ammonia added to different soils on seed germination; cabbage seed 





NH3 

ADDED 


GERMINATION AFTER 






24 
hours 


32 
hours 


48 
hours 


60 
hours 


78" 
hours 


102 
hours 






per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


per cent 




None 


5 


38 


69 


70 


73 


73 


73 




0.02 


6 


30 


64 


70 


71 


73 


73 


Muck ■ 


0.025 


14 


37 


65 


70 


71 


71 


71 




0.05 


8 


32 


61 


65 


68 


70 


71 


• 


0.1 


7 


29 


65 


67 


72 


72 


72 




None 


12 


28 


55 


63 


64 


65 


71 




0.02 


8 


23 


68 


70 


72 


74 


74 


Waukesha silt loam ■ 


0.025 


6 


37 


66 


71 


74 


75 


75 




0.05 


14 


40 


68 


73 


76 


78 


79 




0.1 


1 


15 


47 


63 


67 


71 


73 




None 


13 


32 


60 


70 


73 


80 


82 




0.02 








19 


33 


47 


62 


69 


Quartz sand \ 


0.025 

























0.05 























^ 


0.1 
























TABLE 33 

A ction of various amounts of ammonia added to Waukesha silt loam soil on germination of 

different seeds 





NH3T0 

DRY SOIL 


GERMINATION AFTER 






24 
hours 


32 
hours 


46 
hours 


70 
hours 


94 

hours 


118 
hours 


142 
hours 


166 
hours 


TOTAL 




per cent 


per 
cent 


per 
cent 


per 
cent 


per 
cent 


per 
cent 


per 
cent 


Per 
cent 


per 
cent 


per 
cent 


' 


None 


74 


85 


90 












90 


Lettuce < 


0.04 
0.1 


78 
3 


89 
44 


96 
79 


82 


86 








96 

86 


- 


0.2 































None 


15 


69 


88 


89 










89 


Clover ■. . . \ 


0.04 
0.1 


13 



56 



83 



89 



90 

3 








90 








0.2 




























f 


None 


57 


86 


96 


97 










97 


Rye < 


0.04 


58 


90 


95 












95 


0.1 


16 


70 


91 


92 










92 


- 


0.2 




























r 


None 








12 


35 


56 


63 


67 


68 




Buckwheat ' 


0.04 
0.1 










9 

13 


38 
43 


50 

52 


61 
63 


64 
65 


64 

67 




1 


0.2 





























53 



54 



JAMES JOHNSON 
TABLE 34 



Relative rate of germination of different seeds on soil treated with ammonia; Waukesha silt loam 
treated with 0.05 gm. NH3 {25 cc. of 0.2 per cent NH3 solution) to 50 gm. of soil 









GERMINATION AFTER 








soil, 

TREATMENT 














SEED 


42 
hours 


66 

hours 


90 
hours 


114 
hours 


138 
hours 


162 
hours 


TOTAL 






per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


Wheat \ 


None 
NH3 


55 
51 


97 
97 


98 
•98 








98 




98 


Rye 1 


None 


66 


85 


88 








88 


NH3 


70 


81 


86 








87 


Garden cress i 


None 
NHs 


21 



38 
3 


53 
9 


62 
13 


63 
17 


65 

27 


65 




50 


Bean i 


None 


43 


88 


91 








92 


NH3 


23 


94 


97 








97 


Clover \ 


None 
NHs 


63 
5 


82 
35 


86 
45 


55 


58 




60 


Flax... 1 


None 


98 


99 










99 


NHs 


74 


85 


88 








89 


Cabbage \ 


None 


47 


62 


66 


67 


68 




69 


NHs 


12 


38 


49 


52 


58 




66 


Cucumber \ 


None 
NHs 


41 
8 


84 
76 


85 
82 


84 






85 
85 


Buckwheat \ 


None 
NHs 




8 
4 


25 
21 


36 
28 


54 
40 


60 
50 


63 
61 


Tomato \ 


None 




18 


44 


55 


58 


61 


66 


NHs 




2 


28 


53 


58 


61 


69 


Datura " < 


None 






43 


65 


68 


69 


83 


NHs 






34 


55 


• 57 


60 


97 



by the ordinary method in order to aid further in the correlation of facts (table 
36). From the data it may be seen that at the end of 54 hours and thereafter, 
garden-cress is considerably more susceptible to heated soil than flax seed. 
Where 62 mgm. of ammonia were present, the cress seed was very strikingly 
retarded and flax seed only slightly. A similar proportion apparently exists 
where other strengths of ammonia were used. It is believed, therefore, that 
the results obtained warrant the tentative conclusion that as far as the re- 
sponse of different seeds to the toxic action of ammonia is concerned, a high 
degree of correlation exists between it and the toxic action on these seeds 
produced by heating the soil. 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



55 



TABLE 35 

Relative rate of germination of different seeds on soil treated with ammonia and heated to 250°C* 
Waukesha silt loam 0.075 gm. NHs (25 cc. of 0.3 per cent NHj solution) to 50 gm. soil 



Lettuce . 



Flax. 



Wheat. 



Cabbage . 



Buckwheat . 



Garden cress. 



Clover . 



Tomato . 



Cucumber. 



SOIL 




( 


TERMINATION AFTER 






TREATMENT 


42 
hours 


66 
hours 


90 

hours 


114 
hours 


138 
hours 


162 
hours 






per cent 


percent 


per cent 


per cent 


per cent 


percent 


per cent 


None 


53 


76 


80 


81 






81 


Heated 





1 


10 


18 


23 


39 


58 


Ammonia 


1 


2 


4 


4 


4 


5 


7 


None 


95 












95 


Heated 


91 


96 


97 








97 


Ammonia 





1 










1 


None 


53 


93 


96 








96 


Heated 


49 


91 


96 








96 


Ammonia 


25 


73 


87 


88 


90 




90 


None 


4 


28 


36 


42 


45 


46 


46 


Heated 


6 


3 


6 


8 


18 


27 


44 


Ammonia 











1 


1 


1 


1 


None 


53 


93 


94 


95 






95 


Heated 


50 


90 


93 


94 






94 


Ammonia 


40 


88 


90 


91 






92 


None 


27 


65 


71 


75 


76 




76 


Heated 


12 


26 


29 


30 


38 


53 


65 


Ammonia 























None 


47 


78 


85 








85 


Heated 


3 


21 


42 


50 


61 


71 


80 


Ammonia 





3 


5 








5 


None 





22 


86 


91 


92 


93 


93 


Heated 








21 


71 


86 


88 


92 


Ammonia 








38 


64 


71 


73 


81 


None 





61 


84 


86 


87 




87 


Heated 





55 


82 


87 


87 


89 


89 


Ammonia 





31 


62 


70 


72 


76 


79 



Contained 14.7 mgm. NHs per 100 gm. soil. 



Referring further to table 36, however, it may be seen that in the case of 
both cress and flax greater toxicity to seed germination existed on heated 
soil with only 20 mgm. of ammonia present than was true when 32 mgm. of 
ammonia which had been added to the soil directly were present. The sup- 
position that the difference here is due to the reduced absorptive capacity 



56 



JAMES JOHNSON 



of the heated soil for ammonia seems reasonable. An attempt was made to 
test this theory by adding ammonia to soil heated to 250°C. and 350°C. as 
compared with equal amounts added to unheated soil. For some reason the 
addition of ammonia to soil heated to 250°C. did not increase the toxicity up 
to the expectations; the absorbing power for ammonia was apparently still 
large. On the soil heated to 350°C. however, the absorbing power was greatly 
reduced as indicated by increased toxicity. Unfortunately no further at- 
tempt was made to check up this apparent discrepancy and it must be as- 

TABLE 36 

Germination of seed on Waukesha silt loam soil treated with ammonia as ammonium hydroxide 
and ammonium carbonate as compared with heated soil 





SOIL TREATMENT 


AMMONIA AS 
NIFROGEN 
IN 100 GM. 
SOIL* 


GERMINATION AFTER 




SEED 


30 

hours 


54 
hours 


78 
hours 


102 

hours 


TOTAL 






tngm. 


fer cent 


per cent 


per cent 


percent 


per cent 




None 


3.8 


13 


55 


85 


86 


88 




Heated to 115°C. 


3.8 


11 


46 


67 


72 


84 




Heated to 250°C. 


20.4 





8 


38 


41 


79 




NH4OH 0.1 per centf 


32.0 


2 


16 


58 


70 


71 


Garden cress. . . . ■ 


NH4OH 0.2 per cent 


62.2 





1 


6 


9 


53 




NH4OH 0.3 per eefat 


91.0 



















NH4HCO3 0.1 per cent 


44.0 


1 


10 


46 


69 


88 




NH4HCO3 0.2 per cent 


87.8 



















NH4HCO3 0.3 per cent 


122.4 

















r 


None 


3.8 


77 


96 






97 




Heated to 11 5°C. 


3.8 


61 


94 


97 


98 


98 




Heated to 250°C. 


20.4 


6 


90 


96 




97 




NH4OH 0.1 per centt 


32.0 


72 


94 


95 




96 


Flax ■ 


NH4OH 0.2 per cent 


62.0 


42 


79 


88 


91 


92 




NH4OH 0.3 per cent 


91.0 





4 


7 


8 


8 




NH4HCO3 0.1 per cent 


44.0 


35 


93 


96 




96 




NH4HCO3 0.2 per cent 


87.8 





7 


12 


13 


13 




NH4HCO3 0.3 per cent 


122.4 


















* Represents amount recoverable by magnesium oxide method, 
t Calculated per cent solutions applied. 



sumed that if ammonia is responsible for toxicity in the heated soil, that 
various chemical (61) as well as physical properties of the soil influence its 
manifestation. 

That ammonia exists in the heated soil as an absorbed gas seems improbable. 
Under such conditions it would be gradually lost through areation even in 
dry soil. The toxic property is exceedingly stable, however, in perfectly 
dry soil. The readiness with which it becomes active and is lost in moist 
soils indicates a very unstable condition in the presence of moisture and 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



57 



suggests the similarity of the product to such an unstable salt as ammonium 
carbonate as shown in table 36. For this reason ammonia was added to the 
soil in proportionate amounts both as carbonate and hydroxide. The result 
was practically the same as shown by retarded seed germination. It may 
be assumed that the loss of toxicity in heated soils is purely a chemical ques- 
tion and is due to the breaking down of ammonium salts and the loss of the 
toxic radical as in the case of ammonium carbonate. But assuming further 
that ammonia is actually the toxic agent, a difficulty is at once encountered. 
If ordinary sterilized soils are either naturally or artificially inoculated with 
normal soil flora, the ammonia content as determined by the ordinary meth- 
ods shows a gradual increase, together with a reduced toxicity to seed ger- 
mination. The increased ammonification in sterihzed soils has been repeatedly 
shown by various investigators and has been previously referred to here. 
The results presented in table 37 show that a reduced toxicity to seed germi- 
nation has occurred in the face of increased ammonia content during a period 



TABLE 37 

Germination of lettuce seed on virgin sandy loam after increase in ammonia due to sterilization 

and re-inoculation 



TREATMENT 



Not heated; moist 14 days 

Heated to 115°C.; moist 14 days. . . 

Not heated (kept dry) 

Heated to 115°C.* (just before test) 





GERMINATION 


NITROGEN 


AFTER 


AS NH3 IN 
100 GM. SOIL 




26 


42 




hours 


hours 


mgm. 


per cent 


per cent 


4.7 


92 


99 


11.9 


75 


97 


2.8 


88 


99 


3.7 


11 


94 



per cent 
99 
99 
99 
97 



*Dry. 

of 14 days of exposure to the air. The results indicate a reduced toxicity 
due to, or in close conjunction with bacterial activity favoring ammonifica- 
tion, and experiments were therefore later undertaken to determine in how far 
ammonification, or bacterial activity as such, reduced the toxic action of 
heated soils. 

A theory assuming ammonia to be the toxic agent produced in heated soils 
would appear hopeless here were it not for other evidence. In order to main- 
tain the theory at this point, various phenomena occurring in the fixation of 
ammonia in soil may be resorted to, not only to explain reduced toxicity due 
to changed form, but also to show that these forms of ammonia are easily 
reversible. Assuming that ammonia is the toxic agent, it is possible that it 
may be fixed or changed, chemically, biologically, or physically (43) into less 
toxic but unstable compounds which are again readily broken down to ammonia 
by the relatively violent method of determination of ammonia by distillation 
with magnesium oxide as used in these experiments. On the other hand. 



58 JAMES JOHNSON 

this method may be quite satisfactory for actual ammonia determinations 
in heated soils where such decomposition has already occurred, and is perhaps 
not advanced much further by the distillation process. 

Even though a very satisfactory correlation could be obtained between seed 
germination on soil to which ammonia is added and on heated soil, this would 
not be sufficient ground to prove the relation of ammonia to the toxic action of 
heated soils. The objection can be made that many seeds may react in a simi- 
lar manner to two or more toxic chemical kgents. This subject has been con- 
sidered only in a preUminary way and as far as could be found, the literature 
upon this point is neither extensive nor very helpful. In a preliminary ex- 
periment testing various radically different chemicals such as acids, the strong 
alkalies, various organic bases, and formaldehyde on seed germination, it 
was found that the qualitative differences in reaction were sufficiently great 
in most instances to warrant the conclusion that certain chemicals gave 
fairly distinctive reactions with certain seeds. The qualitative response of 
seeds to ammonia has been previously mentioned. The swollen, blackened 
condition of certain seeds, especially lettuce, exposed to weak strengths of 
ammonia was not in any case approached by exposure to other chemicals 
used. That ammonia causes increased imbibition of water by seeds due 
probably to its action upon the permeability of the seed coat, has been described 
especially by Brown (7) and it is believed that the swelling of seeds treated 
with ammonia in these experiments is due to the same cause. 

In order to get more evidence upon the similarity of the action of heated 
soil, its extracts or distillates, and of ammonia upon seeds, histological 
studies were undertaken. Lettuce seeds which were swollen and killed by 
heated soil extracts and ammonia were fixed with mercuric chloride, im- 
bedded in paraffin, sectioned and stained, and compared with sections of 
normal seeds. In both instances the inner seed coat was found to be greatly 
distended from the cotyledons, which were apparently not greatly affected 
in size. The color change in the seed was seemingly due to the deposition 
of a new substance between the cotyledons and the inner seed coat. This 
substance in sections was of a greenish purple color and seemed to be made 
up of bodies of a fairly definite structure, and usually more or less globular 
in form. Although the study of these sections was not carried out in any 
great detail, the similarity of these bodies to those described by Darwin (11) 
in roots injured by ammonia was especially noticeable and interesting. 

That other substances besides ammonia may react in a similar manner 
with lettuce seed is not questioned but as yet no chemical substance which 
is likely to be present in heated soil, seems to produce this reaction. The 
results on rate of seed germination with formaldehyde as compared with 
ammonia are given in table 38. This may serve to illustrate the point that 
different chemical substances may be expected to behave differently in their 
effect upon the rate of seed germination also. The data indicate that ammo- 
nia at the strength used was relatively more toxic than the heated soil in the 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



59 



case of the four seeds treated. On the other hand, with three seeds formal- 
dehyde was more toxic than ammonia, while in the other case, i.e., the cu- 
cumber seed, it was relatively less toxic than ammonia. This evidence seems 
to illustrate the point that seeds do not on the whole, react in a similar man- 
ner to toxic chemical agents. 

The distinctive odor of heated soils, especially of the products of dry dis- 
tillation, point strongly towards certain organic bases such as pyridine, 
piperedine, or quinoline as being active agents in toxicity to seed 

TABLE 38 
Comparison of the relative rate of germination of different seeds on sterilized soil and soil treated 



with ammonia and formaldehyde; 


Waukesha silt loam heated to 115°C.; 


treated 






with 0.3 per cent NH3 and 1-200 formalin 










SOIL TREATMENT 


GERMINATION AFTER 






42 

hours 


66 
hours 


90 
hours 


114 
hours 


138 
hours 


162 
hours 








per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


per cent 




None 


83 


92 


94 








95 


Lettuce - 


Heated 


8 


40 


67 


83 






84 


Ammonia 


10 


19 


22 


23 


30 




30 


• 


Formaldehyde 





1 


11 


33 


45 


47 


47 


f 


None 


97 


98 










98 


Flax < 


Heated 


92 


94 


95 








95 


Ammonia 


2 


6 


9 


11 


12 


17 


17 


• 


Formaldehyde 






















r 


None 


30 


93 


97 








97 


Wheat " 


Heated 


47 


96 


97 








97 


Ammonia 


18 


93 


98 


99 






99 


• 


Formaldehyde 


8 


69 


95 


98 






98 




None 


5 


65 


82 


84 


85 


86 


86 


Cucumber ' 


Heated 
Ammonia 


10 



71 
31 


86 
76 


87 
83 


88 
84 


86 


88 
86 


^ 


Formaldehyde 





53 


81 


86 


87 


91 


91 



germination or plant growth. In the absence of any satisfactory quaUtative 
or quantitative method of determination of these bases in the presence of 
ammonia, considerable significance can be attached to the qualitative effect 
of ammonia upon lettuce seed as a means of determination. Pyridine, piperi- 
dine, or quinoHne, and other related compounds do not cause the increase in 
pigmentation of lettuce seed characteristic of ammonia, although some may 
produce swelling of the seed. The response of lettuce seed to ammonia and 
to highly toxic products of heated soil are, however, apparently identical. 



60 JAMES JOHNSON 

Relation of soil flora to reduced toxicity 

The most extensive work upon the nature and loss of toxicity of heated 
soils has been that of Pickering (50 — 53). His conception of the toxic property- 
produced in heated soil as concluded in practically all of his papers, is that it 
is organic and nitrogenous in nature and is destroyed by oxidation. Some 
reasons for believing that the disappearance of the toxic principle is not due 
to a simple chemical oxidation process, but that it is due to the activity of soil 
microorganisms have already been mentioned. In order to obtain further data 
upon this subject an experiment was performed in which soil was stored for 
a period of about two months under various conditions as far as aeration, 
moisture, and activity of microorganisms were concerned. The soil was stored 
in wide-mouthed bottles of 3 liters capacity and placed in a horizontal position 
in order to expose as much of the soil surface as possible to the air. These 
bottles were carefully plugged with cotton. Where it was desired to exclude 
air, bottles of only sufficient size to hold the soil were used and these were 
tightly corked and sealed with paraffin. Virgin sandy loam soil was heated to 
200°C., thoroughly mixed, and the equivalent of 600 gm. of air-dried soil added 
to each of 13 containers. In the unheated checks 600 gm. of air-dried soil 
was used. The soils to be moistened were then watered with an equal quan- 
ity of water, plugged as desired and the heated series sterilized in the auto- 
clave for about 1^ hours at 15 pounds pressure. Certain of the soils were 
then inoculated with various microorganisms while others were kept sterile. 
The treatment of each soil, together with the results, are shown in table 39. 
Although the tests were run in triphcates in some instances they are not in- 
cluded in the table. The data on the amount of ammonia present and the 
toxicity to lettuce seed were taken after 55 days of storage. Transfers of 
soil were made to determine the nature of the flora present or of possible 
contamination. The results are believed to be very significant in indicating 
the nature of the loss of the toxicity in particular. It will be noted that the 
initial production of ammonia on heating to 200°C., as shown in the unaltered 
soils, ranges around 13 mgm., three to four times the amount in the normal 
unheated soil. In the presence of microorganisms and moisture, the am- 
monia content has appreciably increased, due to the activity of either bacteria 
or fungi, either in the presence or absence of good aeration. The importance 
of various fungi as ammonifiers in the soil has been well reviewed and de- 
scribed by McLean and Wilson (44). As was to be expected, the microor- 
ganisms had no influence on the dry soil. In the sterile moist soil whether 
in the presence or absence of good aeration, no appreciable change in ammonia 
occurred. Comparing the toxicity to seed germination with the ammonia 
determinations it may be seen that the reduced toxicity is approximately 
directly proportional to the increased content of ammonia. In other words, 
the toxicity has been reduced by the microorganisms where present and active 
as measured by increased ammonification, but has not been reduced appre- 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



61 



ciably in the absence of these organisms, and in the presence of otherwise 
identical conditions. Such a biological explanation of the disappearance of 
toxic compounds from the soil has been recently suggested by Robbins (59). 
In no case, during the time of storage given and at the temperature and other 
environmental conditions afforded, was the toxicity completely reduced. It 
should be remembered, however, that this heated soil represents a much 
higher degree of toxicity than is ordinarily secured in soil sterilization. The 
conditions for free access of air were of course not the best even though a 
cotton-plugged wide-mouthed bottle was used with the soil layer hardly 

TABLE 39 

Influence of various manners of storage of heated soil on ammonia content and rate of seed 
germination; virgin sandy loam heated to 200°C.; stored 55 days; lettuce seed 







TREATMENT 












GERMINATION 


AFTER 






3 












NITROGEN 

AS 
AMMONIA 
IN 100 
GM. SOIL 
















S5 

u 


T3 


V 

t55 


Inoculated with 


a 
.o 

< 


a 
'o 


3„ 

C 3 

tn 


3 
O 

J3 

per 
cent 


i2 

3 
o 

CN 

per 
cent 


3 
O 

per 
cent 


1 

o 

per 
cent 


i2 

§ 

.a 

00 

per 
cent 


i2 

3 
o 
ji 

o 

VO 

per 
cent 


1 

per 
cent 














mm. 


per 
cent 


1 


+ 


+ 




+ 


+ 


+ 


13.9 








2 


26 


52 


80 


82 




3 


+ 


+ 


5 cc. soil extract 


+ 


+ 




20.9 


13 


68 


94 


97 


97 


99 


99 


99 


5 


+ 


+ 


Aspergillus 


+ 


+ 




15.8 





2 


10 


45 


62 


88 


96 


96 


6 


+ 


+ 


Pyronema 


+ 


+ 




19.5 





5 


40 


80 


88 


94 


94 


94 


9 


+ 


+ 




- 


+ 


+ 


13.3 








1 


13 


26 


57 


80 


96 


11 


+ 


+ 


5 cc. soil extract 


- 


+ 




19.8 





5 


11 


25 


39 


79 


81 


95 


7 


+ 


+ 




+ 


- 


+ 


13.2 








3 


12 


23 


48 


88 




8 


+ 


+ 


1 gm. dry soil 


+ 


- 




13.2 








1 


12 


41 


78 


85 


94 


12 


+ 


+ 


1 gm. dry soil 


- 


- 




12.6 








1 


6 


31 


65 


78 


88 


13 


+ 


+ 




— 


- 


+ 


13.6 








2 


7 


35 


54 


79 


92 


14 


— 


— 




+ 


— 




3.5 


89 


98 


99 


100 








100 


17 


— 


— 




+ 


+ 




4.3 


95 


97 


99 


99 








99 


15 


— 


— 




- 


+ 




7.6 


90 


97 


98 


98 








98 


16 


— 


— 




— 


— 




3.6 


84 


93 


95 


97 








97 



more than an inch in thickness at its maximum depth. This does not in- 
fluence the result, however, in showing the comparative importance of bac- 
terial and "mold" activity and aeration alone in reducing toxicity. The least 
loss of toxicity was obtained in the soil kept sterile, aerated, and dry and the 
greatest loss in the soil inoculated with normal soil flora in the presence of 
aeration and moisture. The efficiency of Pyronema in reducing the toxicity 
is especially interesting, and indicates that this fungus so commonly occurring 
on heated soils may serve a very useful purpose in reducing their toxicity to 
higher plants. 

It may be argued again from this experiment that the increase in ammonia 
content together with decreased toxicity is evidence against ammonia having 



62 JAMES JOHNSON 

anything to do with toxicity to seed germination. We may assume, however, 
that ammonia in the presence of microorganisms is fixed or exists in various 
unstable stages of fixation which are no longer toxic but which may again be 
reduced to ammonia on boiling with magnesium oxide, and is hence recorded 
as ammonia present in the soil, though it no longer plays a part in influencing 
seed germination. That various transition products of ammonia are changed 
to ammonia by sterilization has been found by Nikitinsky (45) and others. 

It is evident that the toxic substance produced in heated soils is soluble in 
water. Therefore assuming that ammonia is the toxic agent, more ammonia 
should be found to be soluble in water where its soil content is highest. This 
was found not to be the case, only about 3.3 mgm. being extractable in the 
inoculated aerated moist soil, whereas 4.35 mgm. were found in the sterile 
aerated wet soil. In the inoculated not-aerated moist soil 5.35 mgm. were 
found to be present, this higher amount being due apparently either to re- 
duced loss of ammonia from lack of aeration or to increased denitrification. 

The differences in odor of the soils kept under these various conditions was 
very marked. Where no activity of microorganisms occurred in the heated 
soils the odor was the same as that of the heated soils immediately after heat- 
ing, whether kept dry or moist, aerated or not-aerated. Where microor- 
ganisms had been active in the aerated cultures all the odor of heated soils 
had apparently disappeared, the only detectable odor being similar to that of 
ordinary soil, except in the case of the Pyronema and Aspergillus cultures 
where a "moldy" odor was very evident. In the case of the not-aerated 
moist inoculated cultures, however, a very marked disagreeable odor, similar 
to that of fermenting manure was present, considerable hydrogen sulfide 
probably being present. The action of denitrifiers in the absence of aeration 
no doubt account for this condition. The results in table 39 together with 
other evidences obtained are believed sufficient to justify the conclusion that 
the loss of the toxic substance from heated soils is not a simple matter of oxi- 
dation proceeding even under aseptic conditions as concluded by Pickering, 
but is due rather to the activity of certain bacteria and fungi which become 
reintroduced into sterilized soils. That Pyronema, which is commonly found 
on sterilized soils is very efficient in this respect has been shown and indicates 
that its preference for heated soils over the unheated soils may be one of re- 
lation to ammoniacal nitrogen, just as other fungi, bacteria or green plants 
differ in forms of nitrogen preferred. 

In studying the properties of the toxic or beneficial agent in the heated 
soils, it is evident that many advantages can be secured by separating the 
toxic property as much as possible from the body of the soil itself. This may 
be accomplished by extractions either with water or other solvents, or by 
means of collecting products driven off from the soil upon heating to different 
temperatures. This latter method is essentially dry distillation. It is be- 
lieved that much important evidence secured in the future upon the subject 
of sterilized soils will be obtained through a more careful study of the 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 63 

extracts or dry distillation products of heated soils. Aside from the work of 
Seaver and Clark (68), very little work has been done upon this phase of the 
problem. 

When soil is heated to various temperatures and extracted with water, it 
becomes evident at once that the color of the soil extract increases gradually 
with the temperature of heating up to 250°C., while further heating decreases 
color until practically a colorless extract is obtained from soils heated to 
350°C. This has been found to be true for all the soils used, the color vary- 
ing from a very light tinge of straw color up to a very dark brown wine colored 
liquid at 250°C. The depth of color varies very much for the different soils, 
and is roughly in proportion to their content of organic matter. Peat soil 
extract usually gave the deepest color, whereas extracts of red clay or Norfolk 
sand showed very little color. The color is a fairly good indication of the 
toxicity of the extract to seed germination, though germination does not 
necessarily always decrease with increase in color, i.e., a sUghtly colored so- 
lution may favor seed germination, as for instance, that from certain soils 
heated to 100°C. 

As has already been stated, the fungus development in extracts of heated 
soil usually increases with the temperature up to 250°C., faUing off again at 
higher temperatures, and the extent of fungus growth can to some extent be 
correlated with the color of the soil extract as can toxicity to the growth of 
green plants. These colored solutions are readily decolorized by filtering 
through boneblack or by other decolorizing agents without apparently affect- 
ing their chemical composition. The color is also rapidly lost in the soil, 
due either to reabsorption or destruction, but may be kept quite indefinitely 
as bottled extracts, although some changes appear to occur. 

Closely associated with color in soil is the odor which is generally quite 
closely correlated with the color. The odor varies considerably from different 
soils but is usually quite characteristic, ordinarily being rather pleasant but 
difficult at times to compare with known odors. Seaver and Clark (68) lik- 
ened this odor to that of caramel and again to pyridine. The odor of pyri- 
dine has also been very characteristic in my extracts together with those of 
picohne and quinoline. These odors are, however, far less marked in the 
ordinary extracts than in the products of dry distillation. 

Soil extracts toxic to seed germination, if stored in stoppered bottles, can 
be apparently indefinitely kept without loss of the toxic quality, although 
some change occurs, especially where no attempt is made to keep the ex- 
tracts sterile. Even in sterile extracts, however, some precipitation of ma- 
terial seems to occur from the filtered solutions on standing, it being consid- 
erably greater with some soils than with others. 

Dry distillation of soil 

To get a more concentrated form of the toxic property than that which 
could be obtained by extraction with water, dry distillation was resorted to. 



64 JAMES JOHNSON 

A large bomb holding about 16 pounds of Waukesha silt loam soil was made 
in such a way that it could be placed in a gas oven to be heated and at the 
same time permitting air to be drawn through the soil and passed through 
bottles containing various solutions. The air was drawn through the soil 
and solutions by means of a filter pump. When drawn into water, the prod- 
ucts were found to be toxic to seed germination and slightly acid in character. 
When evaporated to dryness, however, it became evident that the toxic 
property as well as the acidity was lost, indicating their volatile nature. 
Drawing the distillate from heating soil through barium hydroxide or calcium 
hydroxide showed that enormous quantities of carbon dioxide were driven off. 
This suggested that the increased acidity in heated soils might be due to the 
carbonic acid formed, which is partly corroborated by the loss of the acidity 
of the distillate on boihng. On longer heating and at higher temperatures, 
however, the distillate in water becomes colored and strongly alkaline, and 
also takes on the strong pungent odors of picohne, pyridine, or piperidine, 
masking seemingly some traces of odor of ammonia. Qualitative tests for 
ammonia with Nessler's showed strong reaction. Two dry distillations were 
now run with Waukesha silt loam by heating it between 250° and 300°C., 
and drawing the distillate through a weak solution of hydrochloric acid for 
several hours. This distillate was then evaporated to dryness and a dark 
colored salt was secured. It was again taken up with water, decolorized 
with animal charcoal and finally clean white salt was secured, which in one 
case amounted to 3.15 gm. and in the other 3.92 gm. from 16 pounds of soil. 
This was shown to be ammonium chloride by quantitative determination in 
comparison with chemically pure ammonium chloride. The yield of ammonia 
in the extracted salt was very close to that of the known salt. Tests of seed 
germination in the soil after distillation showed it extremely toxic, indicating 
that only a portion of the toxic agent had been withdrawn. The distillate 
in water from long dry distillation at 200° to 300°C. is darker in color than 
that obtained from water extract of heated soils, and has some oily prop- 
erties which, however, apparently disappear on standing. Its toxicity to seed 
germination is very great; most seeds are killed when sown on filter paper 
moistened with the extract. The distillate has been kept in bottles for two 
years without apparently losing much of its toxicity. Fungi do not grow in 
the most toxic of these. 

If this distillate is redistilled and roughly fractionated, we find that a gase- 
ous product is at first rapidily given off at a rather low temperature. This 
gaseous substance is very alkaline and very toxic, and qualitative tests show 
strong reactions for ammonia. The later fractions show less alkalinity, less 
ammonia, and less toxicity. Not all the alkalinity ammonia, or toxicity, 
however, can be removed by boiling, indicating that some stable compounds 
of ammonia or other toxic agents are present. It is also interesting to note 
that, although the first distillates are colorless, they finally become colored, 
as a probable result of other substances going over at low temperatures in 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 65 

addition to ammonia. The characteristic odors also distill over, and this 
fact, of course, argues against drawing any satisfactory conclusion regarding 
the relation of the ammonia present to seed germination. In relation to the 
toxicity of these distillates to seed germination, it should be noted here again 
that the same distinct qualitative reaction occurs as previously described, i.e., 
the seeds placed in contact with it are first discolored, in the case of lettuce 
becoming greenish black, and secondly that the seeds, especially lettuce and 
clover, rapidly swell to often two or three times their original size, frequently 
bursting the seed coat. Similar but much less marked results were secured 
by drawing air through soil heated in the autoclave at 115°C. 

With reference to the effect of ammonia upon seeds as observed in this 
connection, it may be well to mention briefly the observations of others of a 
similar nature. Pickering (51) also noted that on one heated soil (Takoma 
loam) seeds were turned black when placed upon it. Bokorny (4) especially 
has noted the marked toxicity of very small amounts of ammonia upon seeds, he 
having found usually that 0.01 per cent was very harmful. In a later paper 
this author makes a special point of the fact that the injurious nature of am- 
monia is due largely to the ease with which it is able to combine with the cell 
protoplasm. Brown (7) in studying the selective permeability of the covers 
of the seeds of Hordeum vulgare found that the velocity with which water is 
absorbed from solutions of ammonia is remarkable. Similar osmotic rela- 
tions on the part of ammonia have been previously noted by others (49). It 
is believed that the reaction observed by Brown is similar to the one with 
which we have been dealing and explains the swelling of seeds on distillates 
from heated soils. It should be said in passing that the views presented in 
this respect have been partially suggested by Armstrong (2) in some work 
upon stimulation of plant growth. In working with ammonia, he concluded 
that it seems to be the most important active "natural" stimulant, and 
that carbon dioxide is also efifective as a hormone. He finds ammonia stimu- 
lates plant growth in small amounts and that it is toxic in larger amounts. 
Referring to Russell's work (64) and. that of his collaborators, Armstrong 
adds that two powerful hormones are present in sterilized soils, i.e., ammonia 
and carbon dioxide, the latter being produced by increased oxidation. He 
believes that increased growth can be ascribed in large part to these, and 
that retarded growth may be explained by the assumption that they are in 
excess and that the balance is probably a delicate one. 

The Concentration of the soil solution 

The importance of the concentration of the soil solution in heated soils as 
subscribed to especially by Seaver and Clark (68) and Fletcher (18) is such 
that it seemed advisable to obtain further data upon this subject in relation 
to the observed conditions in the experiments carried on by the writer. The 
works of Pickering (51), Seaver and Clark (68), Wilson (79), and Boyoucos 

SOIL SCIENCE, VOL. VII, NO. 1 



66 



JAMES JOHNSON 



(6) and Koch (33) have all estabHshed the fact that increase in concentration 
of the soil solution occurs on heating soils. Their determinations, however, 
were not for the most part carried on with soils heated to sufficiently high 
temperatures to arrive at a maximum concentration, as found on heating to 
250°C. The method used in the determinations reported in this paper have 
been simply that of determining the lowering of the freezing point in soil ex- 
tracts by means of a Beckmann thermometer, Boyoucos's paper (6) de- 
scribing a method of immersing the thermometer bulb directly into the soil 
came after my determinations on soil extracts had been made. The results 
with soil extracts, however, seem to be equally satisfactory where only com- 
parative results are desired. The method of using soil extracts seemed to 

TABLE 40 

Lowering of the freezing point of extracts of different soils heated to different temperatures 







DEPRESSION OF FREEZING POINT AVERAGE 




SOIL 


TREATMENT 


OF DUPLICATE DETERMINATIONS 


AVERAGE 




1 


2 


3 


4 








•c. 


'C. 


"C. 


'C. 


"C. 


■ 


Not heated 


0.019 


0.012 


0.023 


0.026 


0.020 


Muck < 


Heated to 115°C. 


0.031 


0.035 


0.033 




0.033 


• 


Heated to 250°C. 


0.145 


0.189 


0.086 


0.199 


0.155 


r 


Not heated 


0.009 


0.013 


0.020 


0.021 


0.016 


Waukesha silt loam \ 


Heated to 115°C. 


0.016 


0.024 


0.040 


0.026 


0.026 


- 


Heated to 250°C. 


0.032 


0.056 


0.073 


0.079 


0.060 


r 


Not heated 


0.027 


0.013 


0.011 


0.012 


0.016 


Fine sandy loam I 


Heated to 115°C. 


0.034 


0.009 


0.014 


0.030 


0.022 




Heated to 250°C. 


0.074 


0.044 


0.047 


0.052 


0.054 



possess one difficulty, however, namely that although the readings for any 
one series of solutions gave uniform and constant results, they usually did 
not compare well with readings made of other extracts from the same soil. 
Since this variation occurred in the unheated as well as in the heated soil, it 
is apparently not only due to dififerences in temperature of heating or time 
of heating but also to variations in degree of solution. Since certain soils filter 
with much more difficulty than others, especially those unheated or heated to 
a low temperature, force filtering was used in such cases, and it was thought 
that this or a small difference in time of extraction might explain the variation 
in results. This was shown by preHminary experiments, however, appar- 
ently not to be the case. The averaged results are, nevertheless, satisfactory 
for showing the increase in concentration of the soil solutions in different soils 
when heated to different temperatures. That such increased concentration 
is quite marked is shown by the results summarized in table 40. From this 
table as well as from table 41, it may be seen that the concentration of the 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



67 



soil extract from muck soil heated to 250°C. is two or three times as great as 
that of Waukesha silt loam heated to the same temperature. This is not in 
proportion to the relative toxicity to seed germination or to plant growth and 
indicates that concentration apparently has little to do with the retarded 
germination as contended by Fletcher (18) or to toxic action on green plants 
as proposed by Seaver and Clark (68). Reference to table 41 will show a 
gradual increase in lowering of the freezing point in the two soils when heated 

TABLE 41 
Lowering of the freezing point of extracts of soils heated to various temperatures 





DEPRESSION OF FREEZING 


POINT 




Muck soil 


Waukesha silt 
loam I 


Waukesha silt 
loam II 


Not heated 


°c. 

0.012 
0.011 
0.035 

0.124 
0.189 
0.059 
0.052 


"C. 

0.021 
0.020 
0.026 

0.064 
0.079 
0.053 
0.055 


"C. 
0.009 


Heated to 50°C 


0.011 


Heated to 100°C 


0.016 


Heated to 150°C 


0.037 


Heated to 200''C 


0.060 


Heated to 250°C 


0.064 


Heated to 300°C 


0.036 


Heated to 350°C 


0.048 







TABLE 42 

Lowering of the freezing point of different soils on heating to approximately 25(fC. 



SOIL 


DEPRESSION OF FREEZING POINT; 
AVERAGE OF DUPLICATE 
DETERMINATIONS 


NITROGEN AS 

NHa IN 100 GM. 

HEATED SOIL 




Not heated 


Heated 




Peat 

Muck 


"C. 
0.212 
0.020 
0.017 
0.016 
0.018 
0.018 
0.004 


"C. 
0.636 
0.181 
0.082 
0.071 
0.053 
0.031 
0.021 


mgm. 
66.1 
25.9 


Waukesha silt loam 


12.8 


Virgin sandy loam 


9.0 


Fine sandy loam 


10.2 


Red clay 


2.3 


Norfolk sand 


4.4 







to varioois temperatures up to 250°C. followed by a decrease when heated to 
300°C. A slight increase again on heating to 350°C. seems to indicate that 
at this high temperature marked decomposition of inorganic material has 
begun, following the destruction of the soluble organic matter. From the 
data presented in the last tables, there is shown to be a considerable degree of 
correlation between concentration of soil solution and the temperature of 
heating as far as any one soil is concerned, which condition is in turn correlated 
with such other factors as degree of toxicity, favorableness to fungus growth, 



68 



JAMES JOHNSON 



amount of ammonia produced, and color and odor of soil extracts. There 
seems to be no correlation, however, between concentration of soil extracts 
and these factors when different soils are compared. Determinations of the 

TABLE 43 
Temperatures developed in Dewar flasks with germinating wheat in extracts of heated soil 



EXPERI- 


SOLUTIONS 


TEMPERATURE REACHED 


AVERAGE 


MENT 
NUMBER 


At Start 


After 
Iday 


After 
2 days 


After 
3 days 


After 
4 days 


After 
5 days 


PERATURE 



Fine sandy loam extract 



2 



3 < 



Redistilled H2O 
Not heated 
Heated to 115°C. 
Heated to 250°C. 

Redistilled H2O 
Not heated 
Heated to 115°C. 
Heated to 250°C. 

RedistiUed H2O 
Not heated 
Heated to 115°C. 
Heated to 250°C. 



"C. 


'C. 


°C. 


"C. 


°C. 


'C. 


19.1 


29.5 


44.3 


44.8 


41.6 


39.3 


20.4 


28.1 


40.4 


42.0 


40.8 


37.7 


21.4 


31.3 


42.5 


42.6 


41.3 


38.3 


21.2 


26.6 


37.1 


37.8 


35.4 


32.6 


18.2 


35.0 


41.0 


41.8 


39.8 


36.0 


20.0 


31.0 


37.0 


36.5 


33.0 


29.0 


20.7 


33.7 


39.2 


41.7 


39.2 


38.0 


19.3 


26.8 


32.3 


29.6 


25.8 


23.3 


18.5 


25.8 


36.0 


32.0 


29.0 




19.2 


23.5 


25.0 


22.0 


20.6 




19.2 


25.0 


28.1 


24.0 


22.0 




19.2 


23.8 


26.0 


22.8 


21.0 





°c. 

36.4 
34.9 
36.2 
31.8 

35.3 
31.1 
35.6 
26.2 

28.3 
22.1 
23.7 
22.6 



Waukesha silt loam extract 



r 


Redistilled H2O 


18.2 


31.0 


40.2 


42.0 


42.0 


40.0 


35.6 


1 < 


Not heated 


20.0 


21.3 


31.0 


27.5 


24.0 


22.5 


24.4 


Heated to 115°C. 


20.1 


29.0 


34.0 


31.0 


27.0 


24.5 


27.6 




Heated to 250°C. 


20.0 


27.0 


33.1 


30.0 


26.0 


23.2 


26.5 


r 


Redistilled H2O 


18.8 


28.7 


42.2 


44.2 


47.2 


38.8 


36.6 


2 < 


Not heated 


20.0 


25.0 


35.0 


37.0 


34.0 


31.0 


30.3 


Heated to 115°C. 


20.2 


26.5 


38.5 


40.0 


40.5 


36.5 


33.7 




Heated to 250°C. 


20.2 


27.1 


38.0 


39.6 


38.8 


36.2 


33.3 


r 


Redistilled H2O 


18.1 


27.0 


35.2 


32.0 


28.5 




28.1 


3 < 


Not heated 


20.1 


27.4 


29.5 


25.5 


22.5 




25.0 


Heated to 115°C. 


19.0 


26.8 


33.5 


30.1 


26.7 




27.3 




Heated to 250°C. 


19.3 


24.8 


32.3 


28.1 


24.5 




25.8 



concentration of soil extracts were made from seven widely different soils 
heated to 250°C. (table 42). A wide variation in concentration will be noted, 
and although this appears to bear some relation to the amount of ammonia 
produced on heating, it is considerably less correlated with the toxicity of 
these extracts to seed germination. 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 69 

In this connection it may be worth while mentioning briefly another method 
used in studying the behavior of soil extracts, with the hope of arriving at 
some conclusion as to the nature of their action upon seeds. This method 
was essentially that followed by Darsie, Elliott, and Peirce (10) in their study 
of the germination power of seeds in Dewar flasks by means of recording the 
temperature of respiration. A weighed quantity of wheat was placed in ther- 
mos bottles and sterilized with formaldehyde solution, after which equal 
amounts of extracts of unheated soil, soil heated to 115°C. and 250°C. was 
poured in the flasks. Redistilled water was used as checks. The ther- 
mometers were inserted through cotton plugs and into the wheat. The rela- 
tively small temperature differences secured are due largely to the fact that 
wheat, although cheap and convenient for this purpose, is relatively resistant 
to the action of heated soil extracts. If more susceptible seed had been used 
more marked results would no doubt have been secured. Some of the re- 
sults are, however, presented in table 43 and serve to show the regularity 
of the results secured, in addition to corroborating results secured in actual seed 
germination tests. Comparing the average temperatures, it will be at once 
noted that the extract of an unheated soil is considerably less favorable to 
germination than pure water. Heating to 115°C., however, either destroys 
this toxicity in part or stimulates germination. Extracts of soil heated to 
250''C. cause retardation of germination in wheat as measured by rise of tem- 
perature in thermos bottles and compared with the action of extracts of 
unheated soil. 

Relation of toxic and beneficial properties. 

It has been shown in this paper that a fairly marked correlation exists be- 
tween seed germination and the toxic or beneficial action to plant growth 
when the same soil is heated to different temperatures, and that these are 
in turn correlated with the amount of ammonia present, as well as with the 
concentration of the soil solution. There is a strong presumption, therefore, 
that the property which causes simple retardation to plant growth is the 
same as the one which causes retarded seed germination. The property which 
causes "chemical" injury to plant growth, however, appears to be diflierent 
from that causing retarded seed germination. Virgin sandy loam soil heated 
to 115°C. for 160 minutes, though extremely toxic to the growth of tomatoes 
reacted much the same as unheated soil in so far as lettuce or tomato seed ger- 
mination is concerned. It is a difficult matter to prove definitely that the 
toxic agent causing retarded plant growth, and that retarding seed germi- 
nation are the same, although the most reasonable presumption is that they 
are identical. There is, furthermore, seemingly no relation between the re- 
sponse of seeds to the toxic action and that of the early plant growth of any 
one plant. Lettuce which is for instance very susceptible to the toxicity of 
heated soils as far as seed germination is concerned, is relatively resistant 



70 



JAMES JOHNSON 



to the toxic agent during growth. Tomato plants, on the other hand, which 
are relatively susceptible during growth to the toxic "properties, are more 
resistant in germination than lettuce seed. In general it may be said that no 
apparent striking correlation has been found to exist between seed ger- 
mination and plant growth on soil heated to the same temperature. 
Traube (75) in working with acids and narcotics was also unable to find any 
constant relation between injury to germination and growth. In spite of 
this conclusion, however, one cannot fail to note exceptions. The seeds of 
the cereals are notably resistant to the agents toxic to germination, as are 
the cereal plants in their growth. Certain of the heated soils used, especially 
Waukesha silt loam, which is highly toxic to seed germination, is also rela- 
tively toxic to early plant growth, while others, especially fine sandy loam, 
not highly toxic to seed germination, are not very toxic to early plant growth. 

TABLE 44 

Production of ammonia in Waiikesha silt loam soil heated to different temperatures on standing 

moist in greenhouse 





NITROGEN AS NH3 IN 100 GM. DRY SOIL 


TEST FOR 


SOIL TREATMENT 


Initial 
production 


After 18 days 
"naturally 
reinoculated 


After 18 days 

artificially 

reinoculated 


NITRATES AFTER 
18 DAYS 


None 


mgm. 

2.6 
6.0 
9.1 

14.0 
11.0 

5.5 


mgm. 

2.5 

3.2 

4.1 

10.3 

12.1 

13.9 

10.7 

7.3 


mgm. 

2.1 

2.5 

3.9 

9.0 

13.9 

16.7 

13.6 

7.4 


+ 


Heated to 50°C 


+ Strong 


Heated to 100°C 


-f- Strong 

+ 


Heated to 150°C 


Heated to 200°C 


4- 


Heated to 250°C 


+ Weak 


Heated to 300°C 


4- Weak 


Heated to 350°C 


-h Weak 







Regardless of the relation between the agents toxic to seed germination 
and plant growth, however, it may be well to consider the relation of the am- 
monia produced in the soil upon heating and subsequent to heating upon 
plant growth. AU soils upon heating to different temperatures show usually 
an increasing toxicity to growth up to 250°C., which then falls rapidly 
and is practically nil at 350°C. While this is true for early growth, 
the final yields for most plants at least are usually greatest at 250°C., and fall 
ofif gradually at higher or lower temperatures of heating. With these facts 
only in mind, let us consider the ammonia relation in the soil under such condi- 
tions. Reference to table 44 shows a gradual increase in ammonia due to 
heating up to 250°C. but a falling off at higher temperatures. The period 
between the time of heating and the eighteenth day following may be taken as 
the average period during which toxicity to plant growth ordinarily occurs, since 
with many plants recovery from the toxic action may be well on the way after 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 71 

3 weeks, especially at the lower temperatures of heating. Ammonia deter- 
minations were made after 18 days in both soils, in which inoculation with 
normal soil flora was made soon after cooling and where the soil was left to 
become "naturally" inoculated. These results show that after a period of 
18 days, at least, soil heated to different temperatures and standing moist in 
the greenhouse increases rather than decreases in content of ammonia. These 
results were secured with an uncropped soil, and the question may arise as to 
what might be expected to happen on heated soils where crops are grown 
during this period of recovery. In an experiment Waukesha silt loam was 
heated to different temperatures ranging from 50°C. to 350°C. and planted 
to soybeans. It was found that the ammonia content was reduced to the 
greatest extent in the soils heated to the lowest temperatures (50°-150°C.) 
after 3 months. The soil heated to 200°C. and 250°C. retained the highest 
proportioil of ammonia in relation to the amount produced by heating and 
these soils were stiU toxic to soybeans after 3 months and gave the smallest 
relative increase in yield. With radish, however, a more rapid reduction of 
ammonia content was produced, and the final yield was greatest on the soil 
heated to 250°C. The kind of crop grown apparently influences to a con- 
siderable extent the rate with which ammonia is removed from the soil, as 
well as the yield obtained from these soils. It is well known that heating the 
soil, at least to the higher temperatures, tends to reduce the nitrates present 
to very small amounts. Determinations made on Waukesha silt loam and 
muck by the colorimetric method showed that on heating to 250°C. the former 
was reduced from 2.75 mgm. nitrogen per 100 gm. soil to traces only, and in 
the latter cases from 2.28 mgm. to 1.19 mgm. Heating to 350°C. completely 
destroyed all nitrates. Qualitative determinations have shown that in highly 
heated soils increase in nitrates following heating is very slow, although it 
finally may become estabHshed. At any rate it is evident that the increased 
plant growth where it occurs relatively soon after heating soils to 250°-350°C., 
takes place in the presence of a greatly reduced supply of nitrate nitrogen 
but in an increased amount of ammoniacal nitrogen. Assuming ammonia 
to be present in sufficient amounts to be toxic to the early growth of plants, 
it is not difficult to conceive of the rapid recovery of plants resistant to its 
action owing to a decreasing amount (resulting from its use by the plant as 
well as to other changes) which finally reaches an optimum for the soil and 
for the plant, thus resulting in a very rapid increased growth followed by re- 
duced rate of growth in the presence of a nitrogen supply below the optimum. 
For any one soil, the degree of toxicity, the time required for recovery, and 
the final increased yield are, then, largely dependent upon the temperature 
of heating and the susceptibiHty of the plants to the toxic (or beneficial) action. 
That these soils respond to ammonia fertilization has been repeatedly noted 
and is illustrated in table 45 for tomatoes, where retardation was followed by 
marked increase in growth. The addition of ammonia as hydroxide to soils 
in different amounts, may, then, be regulated to such a point as to produce 



72 



JAMES JOHNSON 



an action quite similar to that resulting from heating the soils to various tem- 
peratures up to 250°C. Small amounts of ammonia show no retardation or 
stimulation, larger amounts very decided retardation. But the soils may 
finally recover completely from the injurious action and in the end show a 
markedly increased yield. Ammonium carbonate will produce similar re- 
sults which in turn compare very well with the growth of tomatoes on soil 
heated to different temperatures (plate 7, fig. 2). Similar action may of course 
be obtained by other chemicals capable of being toxic, and later being vola- 
tilized or changed to a point where they may become a stimulant or a plant 
food. No clear case, however, of a response similar to the "chemical injury" 
of plants on heated soils has been obtained with pure chemicals. 

That green plants are able to substitute ammoniacal nitrogen for nitrate 
nitrogen has been well established (26). It has also been shown that different 
plants vary in their ability to use ammoniacal nitrogen. Although the matter 

TABLE 45 

Yield of tomato vines on muck soil treated with different amounts of ammonia as ammonium 

carbonate 



APPROXIMATE 








STRENGTH 








OF AMMONIA 


DRY 


INCREASE 


EARLY GROWTH 


SOLUTION ADDED 


WEIGHT 






TO 2 KILOS SOIL 










gm. 


per cent 




None 


3.00 






. 1 per cent 


8.75 


191 


Best; no retardation noted 


0.2 per cent 


13.30 


243 


Slight retardation 


0.3 per cent 


12.10 


303 


Marked retardation 


0.4 per cent 


11.25 


275 


First plants killed; reset; marked retardation 



has not been investigated from this point of view, it seems reasonable to 
suppose that the differences in plants in their response to soil sterilization 
may be due in part to their capability of using ammoniacal nitrogen in the 
absence of nitrate nitrogen. 

In this connection a study of the growth of plants on heated soils under 
aseptic conditions was made in order to determine the relation of the soil flora 
to the supply of plant food. Experiments in growing plants under sterile 
conditions in heated soil were started. Various forms of apparatus hayebeen 
devised by investigators for growing plants under aseptic conditions, but 
these are ordinarily too complicated to permit the use of any considerable 
number of such structures. "Kellerman culture chambers" were finally 
selected for use in the experiments recorded here. These proved very sat- 
isfactory excepting for the fact that aeration is very poor under the condi- 
tions there afforded. It was found, therefore, that when a soil relatively 
toxic was heated or when plants relatively susceptible to toxic 
action were used, the time required for the plants to recover from the 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 



73 



injurious action was too long to permit a study of the beneficial action 
of the heated sterile soil on plant growth. Finally by selecting 
the least toxic soil (fine sandy loam), heating to 115°C. and planting to wheat, 
which is relatively resistant to the toxic action, it was found that fairly re- 
liable results could be secured. That sterile conditions were obtained was 
demonstrated by transferring small bits of this soil with a sterile needle into 
bouillon tubes just before harvesting the crop. In the last experiment, the 
results of which are presented in table 46, one of the three flasks supposedly 
sterile had become infested with PeniciUium but was apparently sterile inso- 
far as bacteria were concerned. The increased growth on the reinoculated 
soil above that of the checks and sterile cultures is suflacient to be of consid- 
erable significance as indicating the relation of bacterial activity to the bene- 



TABLE 46 

Growth of wheat under sterile conditions as compared ivith unsterilized soil and sterilized and 

reinoculated soil 



TREATMENT 


NUMBER 


DRY WEIGHT 


AVERAGE 


INCREASE 


No treatment \ 


1 
2 
3 

4 
5 
6 

7 
8 
9 


gm. 
0.160 
0.179 
0.160 

0.226 

0.216 

* 

0.515 
0.335 
0.467 


gm. 
0.166 
0.221 

0.439 


per cent 


Sterile soil i 


32.2 


Sterilized and reinoculated i 






164.4 



* This jar became accidentally infested with a fungus (PeniciUium) and the yield was 
0.482 gm. 

ficial action of heated soils. The explanation of the results secured is not 
as simple as it may appear at first sight. Ordinarily it would be concluded 
that the increased growth on the reinoculated sterile soils is due almost en- 
tirely to increased bacterial activity in the sterilized soil as a result of new 
conditions which make this increased activity possible. The object of this 
experiment was to determine the relative importance of bacterial activity as 
compared with the direct chemical action of heat on soil in rendering food 
available for plants. It seems reasonable to assume that much of the in- 
creased plant growth on heated soils (especially of those heated to high tem- 
peratures) is due to direct chemical action in rendering plant food available. 
The results secured in growing plants under sterile conditions point quite 
strongly in this direction, since it was found that the plants grown in the 
sterile soil made an appreciable increase above the unheated checks in spite 



74 JAMES JOHNSON 

of a toxic action sufficient to retard greatly the proper functioning of the root 
system. This condition was shown by the root systems in the sterile cultures 
which were very short, thick, small in total area, lacking in root hairs, pene- 
trating only to shallow depths in the soil and generally unhealthy in 
appearance. 

In the sterilized soils where reinoculation was made, the toxicity of the 
soil was apparently rapidly destroyed. The root system benefited thereby 
in quite a striking manner, being considerably better than that in the sterile 
checks or in the unheated soil (plate 8, fig. 2). Since all other factors were 
the same in the flasks except for the presence of soil organisms, it is evident 
that the destruction of the toxic substance was due in a large measure if not 
entirely, to their action. It seems extremely probable, therefore, that the 
increased growth in the reinoculated sterilized soil as compared with the 
sterile soil, was due in some considerable measure to the destruction of the 
toxic substance, which retarded the growth in sterile cultures but which was 
relatively rapidly destroyed or lost, or perhaps, even converted to plant food 
in the reinoculated soil cultures, and that the smaller growth in the sterile 
cultures was due as much to the toxic agent as to lack of increased food 
supply due to bacterial activity. This experiment illustrates further 
the important part played by soil organisms in benefiting sterilized soils due 
to the destruction of the toxic agent, making it possible for the plants to bene- 
fit sooner from the plant food rendered available as a result of the direct and 
indirect action of the heat upon the soil. 

Conditions favoring development of microorganisms in heated soil 

Turning to a consideration of the growth of fungi on heated soil, two pos- 
sible hypotheses suggest themselves in the broadest sense. These are that a 
condition or substance unfavorable to fungus growth exists naturally in soil 
and is destroyed upon heating, or that new conditions or substances favorable 
to fungus growth are produced in heated soils. The former theory is essen- 
tially that of Kosaroff (35) who supposed that the soil contained certain 
toxins for fungi which are destroyed upon heating. Seaver and Clark (67, 68) 
have done a considerable amount of work in the support of the latter theory 
and conclude that the increased concentration of the soil solution favors fun- 
gus growth, which may at the same time be retarding in its influence upon 
the growth of green plants. Kosaroff 's theory rests primarily upon the fact 
that the addition of the extract of a heated soil to an unheated soil will not 
permit of the growth of Pyronema upon the unheated soil. Seaver and Clark 
also obtained the same result. The writer was able, however, to obtain a 
good growth of Pyronema upon unheated soil by the addition of a heated 
soil extract following sterilization of the unheated soil by means of formalde- 
hyde. It is highly improbable that a formalin drench (1-50) destroyed the 
soil toxin (and if it did it alone should permit of the growth of Pyronema), 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 75 

but it did quite satisfactorily sterilize the soil for a time. It seems evident 
in view of the results previously described on the relation of the activity of 
soil microorganisms to the reduction of the toxic products produced in heated 
soil, that extracts of heated soils added to unheated soils are changed in 
character before the growth of Pyronema can establish itself. Although the 
formalin may have slightly increased the concentration of the soil solution 
(33) it is believed that its sterilizing properties were responsible for the changes 
produced which permitted the growth of Pyronema on heated soil extract 
added to unheated soil. This observation together with the findings of 
Seaver and Clark are believed sufficient to overthrow Kosaroff's arguments 
on the relation of soil toxins to the development of Pyronema. 

That the concentration of food material as such plays an important part 
in the growth of fungi on heated soil as suggested by Seaver and Clark 
(67) seems doubtful in view of the following facts: the concentration of 
certain heated soils on which Pyronema grows readily may be considerably 
less than that of certain unheated soils or soils heated to 350°C. on which 
Pyronema will not establish itself. This is illustrated by the fact that Py- 
ronema will make a good growth upon heated red clay or Norfolk sand or 
even relatively pure sand in which the concentration of the soil solution is 
relatively very low as compared with that of unheated peat or muck soil upon 
which Pyronema will not grow. Again the disappearance of the favorable 
condition for Pyronema from the soil seems to be considerably more rapid than 
the loss of concentration. At least, it is not to be expected that the food sup- 
ply for the fungus is so rapidly diminished as to materially check the fungus 
growth after a period of only a few days. This statment needs of course, to 
be supported by the observation that more than one "crop" of fungus growth 
can occur upon any one heated soil. 

That soils steriHzed by heat at low temperatures or by antiseptics become a 
favorable medium for subsequent bacterial development has been shown 
repeatedly by various investigators beginning with the work of Hiltner and 
Stormer (25) . The principal point of argument in recent years as to the effect of 
sterilization on the soil has been centered around the explanation of the cause 
of this increased bacterial activity. On account of the seemingly close cor- 
relation between the rate of bacterial development and of fungus growth on 
soils heated to different temperatures there apparently is no reason for dis- 
tinguishing between the cause of the rapid development of these closely 
related microorganisms on sterihzed soils. If this be true, then Russell's 
protozoan theory (64) would necessarily have to extend the activity of the 
phagocytic organisms to the limitation of fungus growth as well as to the 
Hmitation of bacterial development. The special favorableness which Py- 
ronema finds for growth upon heated soils over that of other fungi is, how- 
ever, apparently a matter for discussion aside from that of ordinary increased 
development of microorganisms in heated soils. The arguments of Seaver 
and Clark are apparently not concerned with this fact, but, using Pyronema 



76 JAMES JOHNSON 

as an example, they attempt to explain why fungi in general find the soil a 
more favorable medium for growth. On the same assumption as previously 
made their conclusions might then be made to apply also to the explanation 
of increased bacterial activity in soil. The subject then finally resolves itself 
into two separate problems, (a) Why do microorganisms in general find a 
sterilized soil a more favorable medium for development? (b) Why does 
Pyronema confluens in particular find heated soils a more favorable medium 
for growth than do other fungi? The first problem will be dismissed with 
the simple statement that the writer's observations on a wide variety and type 
of heat sterilized soils have led to the opinion that in practically all cases the 
increased activity of microorganisms in sterilized soil can be explained on 
the basis of reduced competition (a wide variety of organisms are known to be 
negatively " chemotropic" to each other) in the presence of increased food 
supply without any special reference to the concentration of this food supply 
as such. 

Seaver and Clark (68) assume as one basis for their theory on the growth 
of Pyronema in heated soil, that a concentration of soil solution favorable to 
fungi is unfavorable to green plants. The experiments of Raulin (57), and 
especially Bauman (3) who noted the relation of zinc salts to plants, and 
found that fungi are able to grow in solutions of zinc exceedingly toxic to 
higher plants, and Heald (23) who found the toxicity of carbolic and other 
acids to green plants greater than to fungi, illustrate the fact that certain 
fungi are relatively resistant to toxic substances. It is now generally rec- 
ognized that the resistance of fungi to toxic agents is greater than that of 
green plants. It is not surprising, therefore, that fungi should grow on heated 
soils highly toxic to green plants regardless of the concentration of the soil 
solution, providing that these fungi find the substratum congenial because 
of reduced competition and increased food supply of either a general or a 
special nature. If we now reason a step further we should expect to discover 
that some fungi will find heated soils more favorable for growth than others, 
due to a modified food supply, as shown by Nikitinsky (45) and others, in 
the same way that certain green plants find heated soils more favorable for 
growth than others do since fungi as well as green plants differ in their food 
requirements. The fact remains, however that Pyronema is capable of re- 
ducing the toxicity to seed germination (and probably also to plant growth,) 
while growing on heated soil and at the same time of increasing the ammonia 
content of the soil. What relation the compound favorable to the growth 
of Pyronema has to the substance toxic to seed germination must, however, 
still remain obscure. The presumption is all in favor of it being a nitrogenous 
compound, but no good evidence exists on this point. 

DISCUSSION OF RESULTS 

Some of the conclusions drawn from the data presented in this paper have 
already been pubHshed in a preliminary paper (28). More data to support 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 77 

the conclusions drawn have been secured since the pubHcation of this first 
paper. On the other hand, some contradictory evidence on one point in 
particular has been obtained. That the instability of ammonium carbonates 
in heated soils, except when kept in a dry and unaerated condition, accounts 
for the gradual decrease of the injurious action is not supported by all the 
facts, since destruction of the carbonate was attributed to purely chemical or 
physical phenomena. The results secured since have shown the importance 
of microorganisms in the loss of toxicity. That the reduced toxicity is due 
to biological action upon ammoAium carbonate, resulting in loss of gaseous 
ammonia, or to its fixation in the soil seems now most probable. 

It seems evident from these studies that a large number of difficulties pres- 
ent themselves in the way of obtaining direct evidence upon the toxic action 
of the ammonia produced in heated soils. It has been felt in the course of 
the work that sufficient emphasis has not been placed upon quantitative de- 
terminations of the ammonia present, as such, for comparison with the toxicity 
of similar amounts in and out of the soil. On the other hand such evidence 
has seemed of minor value in view of the extraordinary influence of various 
chemical, biological, and physical processes in the soil on the ammonia rela- 
tions. The extensive literature upon ammonia relations in soil abounds with 
evidence of the great variability of results secured in fertilization, ammoni- 
fication, fixation, and absorption experiments. It seems worth while, how- 
ever, to go to the literature to find confirmatory evidence for some of the 
conclusions presented in this paper. 

With reference to toxicity of ammonia in soils to plant growth under con- 
ditions where it is reasonable to assume the amounts present are no greater 
than those produced in heated soils, mention may be made of the observa- 
tions and experiments of Pitsch (54), Wagner (76), and Ehrenberg (14). 
Other investigators who have dealt with the susceptibihty of seeds and plants 
to injury from ammonia in soils are Sigmund (69), Coupin (9), Ehrenberg 
(15), and Bokorny (4). Especially interesting in this connection are some of 
the results secured by Sigmund (69) and Ehrenberg (15) on the influence of 
zinc on soil. It has been shown that zinc is able to liberate ammonia from 
ammonium salts, which is then said to act corrosively on the plant roots 
through its hydroxyl ion, but on account of its easy dissociation, the am- 
monia partly volatilizes. Sterilization of the soil increases the injury while 
the action of the nitrate-forming organisms in removing ammonia compounds 
results in lessened or prevented injury. It appears, therefore, that we have 
in the action of zinc on soil a condition similar in many respects to that of 
heated soils. 

As early as 1880, a very fundamental study was made by Nivet (46) on 
the relation of ammonia in the soil. This investigator found that ammonia 
occurs normally in soils as ammonium carbonate, and that the addition of 
ammonia in form of sulfate, results in ammonium carbonate being produced 
through its reaction with the calcium carbonate. The ammonium carbonate 



78 JAMES JOHNSON 

« 

being more or less unstable, considerable loss of ammonia from the soil may 
result, which loss varies greatly, however, with the nature of the soil. A 
great variation in the absorptive property of the soil was also noted in this 
connection, it being found that the ammonia given off from sandy soil was 
seven times as great as that from "humus" soil. In the presence of a con- 
siderable excess of pure carbon dioxide, the loss of ammonia by volatiliza- 
tion was reduced to an inappreciable amount. From the results of the in- 
vestigations of Nivet (46) and others, it is evident that a consideration of the 
toxicity of ammonia in soils involves to a considerable extent its relations to 
the absorptive capacity present. The phenomena of absorption in soils has 
been known for nearly a century and many of the early workers as Way (77) 
had a good conception of the part played by this factor in heated soils, but 
more fundamental evidence from our standpoint has been brought together 
by recent workers, especially Richter (58), Wagner (76), Stoklasa (72), Pres- 
cott (56), and Cook (8). The most important facts advanced by these writers 
are as follows. Soils have a remarkable degree of power for absorbing gases, 
and especially ammonia, but they differ very widely in this capacity. In 
general the heavy types of soil, or those high in humus or vegetable matter, 
have a high absorptive capacity, and those of a light nature or low in vege- 
table matter have a low absorptive capacity. A striking variation from this 
rule, however, may be expected, and the actual or relative absorption of any 
soil can only be determined by actual trial. Absorption of ammonia is not, 
however, a simple physical process, but chemical and colloidal properties are 
also involved, together with many exterior influences, such as the tempera- 
ture and moisture content of the soil. Chemical absorption may, for in- 
stance, result in the formation of an insoluble chemical compound. Colloidal 
matter increases the power of absorption. Calcium, especially calcium oxide, 
increases the absorptive power for ammonia in a decided way. Heating the 
soil decreases the absorption or fixation, a fact probably due largely to the 
action on humus and colloidal matter. High temperatures are generally un- 
favorable and low temperatures favorable to absorption, while increased 
moisture supply tends to permit of greater absorption. 

With these facts in mind, it may be assumed that the following approxi- 
mates the conditions in soil heated to 250°C. The maximum ammonia con- 
tent is present in connection with a reduction in absorptive capacity of the 
soil. The amount of the measurable toxicity is then the result of the bal- 
ance between the total ammonia produced on heating and the total absorp- 
tive capacity which exists in the heated soil. Heated soils with high ammonia 
content but also with high absorptive capacity, as is true in the case of peat, 
are therefore, less toxic than Waukesha silt loam with lower ammonia con- 
tent, but with much lower absorptive capacity. Theoretically, a correlation 
should exist between the ammonia content and the absorptive capacity on 
the one hand and the toxicity to seed germination and plant growth on the 
other. The presence of numerous other disturbing factors renders such an 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 79 

expectation practically unobtainable. The studies of Kanda (29) on the 
stimulatory action of metallic salts upon plant growth are merely cited to 
show the importance of such disturbing factors. In the case of copper sul- 
fate, for instance, Kanda found it was difficult to get constant results in ex- 
periments because of the influence of such factors as the action of the salt upon 
the humus compounds of the soil, the influence of the time of the year, and 
especially temperature and moisture changes in the atmosphere. It was 
found that in a cold, moist atmosphere, the toxic action was much less than in 
a warm, dry atmosphere. Other meteorological and cHmatological factors 
were also found to influence results. In a similar manner, the influence of 
environmental factors, some of which are as yet not clearly understood, ap- 
pear to complicate the data secured on seed germination and plant growth on 
heated soils. 

The action of ammonia or ammonium compounds as plant food has been dem- 
onstrated (26). Lyon and Bizzell (42), Kelley and McGeorge (30) and others, 
as well as results of the author have also shown that nitrates are reduced in 
heated soils and that they are practically non-existent on soil heated to 250°C. 
Furthermore, according to some writers, heated soils become unfavorable for 
bacterial activity and although this conclusion is doubtful in all cases, it is at 
least probable that it takes some time before nitrification can reestablish 
itself to a decided extent. In the absence of nitrates, but with large supplies 
of ammonia present, it is probable that ammonia is being used directly by the 
plant as a source of nitrogen where plant growth occurs. That cereals in the 
presence of ammonia at least could develop in soils free from nitrates was con- 
cluded by Pitsch (54) as early as 1887. The researches of Kossowitsch (36) 
and Hutchinson and Miller (26) have added sufficient eviaence to make it 
appear unquestionable that agricultural plants of various kinds can produce 
normal growth when supplied with nitrogen as ammonia in the absence of 
nitrates. 

There is apparently room for difference of opinion as to the form of ammonia 
existing in heated soils. Ammonia as such, probably is never present in nor- 
mal soils. When ammonium salts are added to soils, it is generally believed 
that ihey pass through several stages of decomposition and nitrification be- 
fore being taken up by the plants. It is known that under ordinary conditions 
much ammonia exists in the soil as ammonium carbonate. On the other 
hand, it also appears that some of the ammonia enters into very stable com- 
binations, from which it is no more readily separable (61). Eliminating for 
the present the biological factor in the soil, such as may be expected to occur 
in soils while heating or in highly heated soils stored dry, one is confronted with 
a chemical or at most a physico-chemical problem. There appears to be 
several reasons for the assumption that much of the ammonia produced in 
heated soils exists as some form of carbonate of ammonia. These are briefly: 
(a) The high production of carbon dixoide coincident with that of ammonia 
production may be expected to result in the combination of the two when cool- 



80 JAMES JOHNSON 

ing to lower temperatures, (b) The stability of the high ammonia content 
in the soil when kept dry in the presence of reduced absorptive capacity ar- 
gues against its existence as a gas. (c) The comparative ease with which 
ammonia is driven off from a soil extract at low temperatures indicates that 
it exists as an unstable compound, (d) The slight increase of acidity in 
heated soils indicates an excess of an acid radical, most probably carbon diox- 
ide, which is produced in large amounts on heating, and hence reduces the 
possibility of ammonia remaining in the soil for any length of time as a free 
base. 

When heated soils are kept moist and aerated, the carbonate of ammonia 
present is no doubt gradually dissociated into ammonia and carbon dioxide, 
only to be reabsorbed by the soil, changed or fixed into other forms by biolog-. 
ical or other action, or possibly lost in small amounts to the atmosphere, de- 
pending upon the favorableness of the soil for the activities concerned. These 
changes in heated soils together with the ammonia removed by growing plants, 
where present, explain the loss of ammonia from heated soils and hence may 
account for reduced toxicity. As a matter of fact, however, an increase in 
ammonia actually occurs for a period of time following sterilization of the soil 
in spite of gradually reduced toxicity. Here the assumption must be made 
that the ammonia exists at this critical stage, not as toxic gas, but in various 
deHcate stages of transition between humus compounds and ammona on the 
one hand and between ammonia and nitrates on the other, and that these 
compounds are reduced to ammonia in the determinations of total ammonia 
present in the soil. In this process of transition, microorganisms play the 
main role, and we have shown that microorganisms are largely responsible 
for the destruction of the toxic property in the soil. It is of course equally 
probable that certain organic compounds, which may or may not be transition 
products of ammonia, and which result from decomposition of humus on 
heating, may be the toxic property involved. In contradiction of this we have 
only the evidence of the "qualitative" similarity of the action of ammonia 
and its compounds on seed germination as compared with that of highly toxic 
heated soils or the extracts of such heated soils. This evidence, together with 
the fact that ammonia or ammonium salts added to soils in correct amounts 
may produce marked similarity in action to that produced by heated soil on 
seed germination and plant growth, constitutes the main evidence for the con- 
clusion that ammonia plays a large role in both the toxic and beneficial action 
of heated soils to seed germination and plant growth, although it may not be 
the only factor concerned. That all seeds and plants do not respond in a 
similar manner to heated soils is believed to be due to the difference in the 
selective permeability of the seed coat or of the plasma membrane to the toxic 
agents. This may also explain the ability of certain plants to assimilate 
nitrogen in the form of ammonia or ammonium salts either in the absence of, or 
in a reduced supply of other forms of nitrogen and hence may account in part 
for the beneficial action of heated soils on certain plants while they appear at 
the same time to be injurious to other species of plants. 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 81 

SUMMARY 

1. The purpose of this investigation has been primarily to arrive at some 
conclusions as to the nature of the action of sterilized soils upon plant growth, 
in order that their use in soil biological and phytopathological research as well 
as in prophylactic measures may be more clearly understood and more pro- 
ductive of rehable results. 

2. Practically all soils when sterilized in the ordinary manner by heat at 
temperatures approximating 100-1 15°C. produce temporary retardation to 
seed germination and plant growth followed by increased rate of growth. The 
extent of this action varies very greatly with the soil, seed, and plants used 
and with the environmental conditions existing. To study the factors con- 
cerned, 7 different soils were subjected to widely varying treatments and con- 
ditions, and their action on various seeds and plants determined. Although 
some of the results secured are of a corroboratory nature, these have been ex- 
tended or limited in their application. 

3. On heating a soil to different temperatures, it was usually found that a 
gradual increase in toxicity occurs to seed germination and to early plant 
growth which reaches its maximum at approximately 250°C., but gradually 
decreases to practically no toxicity on soils heated to 350°C. or above. 

4. The time required for recovery from this toxic action is usually directly 
proportional to the intensity of toxicity produced, but the final beneficial ac- 
tion is often greatest on the soils exhibiting the greatest injurious action on 
early plant growth. 

5. Different soils vary markedly in their behavior upon heating to the same 
temperatures both in toxicity and in beneficial action to seeds and plants, 
and this is not seemingly correlated with any single distinguishing character 
in the soil, but is correlated rather with the balance of all the factors 
concerned. 

6. Seeds vary greatly in their sensitiveness to the toxic action. Lettuce 
and clover seed are for instance, very susceptible to the toxic substance, 
whereas seeds of wheat, buckwheat or flax are very resistant. The degree of 
sensitiveness of seeds is roughly characteristic of their genetic relationship. 
The Gramineae and the Cucubitaceae are usually resistant and the Legumi- 
noseae and Solanaceae are, as far as determined, more susceptible. 

7. With the seeds resistant to the toxic action marked acceleration of rate 
of germination may occur on even highly heated soils. This is no doubt an- 
other expression of the same substances that cause retardation in more sus- 
ceptible seeds. Seeds classed as susceptible may on the other hand show ac- 
celerated germination on soils not productive of high toxicity on heating or 
upon heating soils to low temperatures only. 

8. Growing plants differ markedly in their sensitiveness to the action of 
heated soils. Soils very toxic to certain plants, such as tomatoes, may be very 
beneficial to others such as wheat. The similarity of the behavior of growing 

SOIL SCIENCE, VOL. VII, NO. 1 



82 JAMES JOHNSON 

plants and germinating seeds in this respect suggests that the injurious and 
beneficial substance in both cases may be the same. Toxicity to seed germina- 
tion, however, is seemingly not always correlated with toxicity to plant growth 
and vice versa. Furthermore, sensitiveness of seeds to the toxic agent in ger- 
mination is not indicative of the behavior of the same species in its growth on 
the same soil. 

9. The growth of fungi on heated soil is correlated with the toxicity to 
seed germination and plant growth on any one soil. The growth of Pyronema 
especially has been studied. This and other fungi and apparently bacteria 
also grow best in soil heated to 250°C., diminishing in rate and profuseness 
of growth at lower or higher temperatures of heating. 

10. The ammonia content of a soil heated to different temperatures is 
highest on heating to about 250°C. and diminishes gradually at higher and lower 
temperatures of heating. This is also true for the concentration of the soil 
solution. Ammonia content and concentration of the soil solution are there- 
fore roughly correlated with the degree of toxicity to seed germination and 
early plant growth and the extent of the beneficial action to late growth of 
green plants and to growth of lower microorganisms in any one soil. 

11. There is apparently, however, no correlation between these factors 
when different soils are compared to each other. The toxicity of heated 
Waukesha silt loam with a relatively low ammonia content is, for instance, 
much greater than that of heated peat with a relatively much higher ammonia 
content. The absorptive capacity of the two soils is, however, vastly different, 
and this in turn markedly influences the action of toxic compounds produced 
in the soil. In water extracts of heated soils, the toxicity to seed germination 
is more directly proportional to the ammonia content. It seems that the 
toxicity of the soil is, therefore, not only determined by the amount of the 
toxic agent produced by heating, but also by the absorptive capacity of the 
soil for the toxic agent as well as by a number of other factors, the additive 
and subtractive value of which gives a balance of toxicity very difficult to 
properly analyze. 

12. The addition of ammonia as such to soil in varying amounts may be 
made to produce a condition similar in many respects to that produced by 
heated soils on seed germination and plant growth. 

13. Very similar "qualitative" reactions can be produced with certain seeds 
on highly toxic heated soils or their extracts and with certain strengths of 
ammonia. These reactions do not seem to be reproducible with chemicals 
other than ammonia or ammonium salts. 

14. It is beUeved that much of the toxic action in heated soils is due to the 
ammonia produced and that it exists in the heated dry soils largely as am- 
monium carbonate, which is, however, gradually decomposed under normal 
growing conditions. 

15. All the toxic properties in heated soils are not, however, believed to 
be the same. Certain changes termed "chemical" injuries are seemingly 
due to quite different causes than ordinary retardation. 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 83 

16. The toxic property is volatile and is destroyed or changed to non-toxic 
compounds in soils kept under normal growing conditions. This has been 
shown to be due to the activity of ordinary soil flora, which, however, may 
apparently at the same time increase the amount of ammonia present. 
Pyronema confluens has also been shown to be efficient in both these respects. 
The reduced toxicity in the presence of increased ammonia content in the soil 
is believed to be explained by its existence in various dehcate transition stages 
rather than as true ammonia due to the activity of soil organisms and that 
these transition products are broken down when the ammonia determinations 
are made. The reduction of the toxic property to seed germination and plant 
growth by the activity of soil flora has been shown and is contrary to Picker- 
ing's conclusion that loss of toxicity in storage of heated soils is an oxidation 
process which may go on under aseptic conditions. 

17. The beneficial action of highly heated soils is believed to be due in a 
considerable measure to the ammonia liberated on heating, since increased 
growth may result in almost total absence of nitrates or in heated soil under 
aseptic conditions in spite of a considerable toxic action upon the roots. The 
fact that certain green plants are able to take up their nitrogen in the form of 
ammonia is believed partially to explain the variation of sensitiveness of 
plants to heated soils. 

18. The temperature of the soil is an important factor in determining the 
extent of the toxic and beneficial action to plant growth. The toxic action 
disappears more slowly and is more destructive at low soil temperatures (be- 
low about 25°C.) than at higher temperatures. 

19. Observations on the growth of Pyronema seem to indicate that the fav- 
orableness of heated soils to its growth is not entirely one of concentration of 
soil solution, as argued by Seaver and Clark. Pyronema will grow on heated 
soils very low in concentration as compared with other unheated soils on which 
growth never occurs. All microorganisms appear to grow better on the soils 
of higher concentration of soil solution, owing to increased food supply, but 
the type of organism and the extent of its growth will vary with the com- 
petition at hand and the kind of food materials present. Fungi differ in their 
food requirements in much the same way as green plants, and heating the soil 
no doubt produces chemical substances especially favorable to the growth of 
Pyronema. 

20. The conclusions drawn here are considered to apply particularly to 
highly heated soils, although it is not believed that any fundamental difference 
exists between ordinary steam sterilized soils and highly heated soils rein- 
oculated with normal soil flora. 

21. Although the injurious action of heated soils on plant growth has been 
brought into the foreground in this paper, it is not desired to leave the impres- 
sion that heat sterilized soils are of questionable value in research and practice. 
The opposite is rather true and little hesitancy need be felt in recommending 
steam sterilization of soil for practical purposes or for use in research problems 



84 JAMES JOHNSON 

where it is necessary to eliminate certain organisms from the soil. But, one 
must be prepared to expect a short period of retardation of growth followed 
by a beneficial action, and in special cases, with certain soils or plants or under 
certain environmental conditions, a very marked interference with the nor- 
mal development of plants. 

In conclusion, the writer wishes to express his appreciation of the helpful 
suggestions given by several colleagues at the University of Wisconsin dur- 
ing the progress of this work. 

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PLATE 1 
The Influence of Heated Soils on Seed Germination and Plant Growth 

Fig. 1. Showing the difference in response of different soils on heating to 110°C. on the 
growth of tomatoes. A, Check, soil not heated; B, heated soil. Pots lA-lB, Miami silt 
loam; pots 2A-2B, muck; pots 3A-3B, red clay; pots 4A-4B, greenhouse compost; pots 5A- 
5B, peat; pots 6A-6B, virgin sandy loam. 

Fig. 2. Effect of heating virgin sandy loam to different temperatures, showing the retard- 
ing influence on plant growth. 1, Not heated; 2, heated to 50°C.; 3, 100°C.; 4, 150°C.; 5, 
200°C.; 6, 250°C.; 7, 300°C.; S, 350°C. 

Fig. 3. Growth of radish on muck soil heated to different temperatures illustrating the 
beneficial action to late plant growth. A, Not heated; B, heated to 115°C.; C, heated to 
250°C.; D, heated to 350°C. 



88 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 

JAMES JOHNSON 



PLATE 1 




Fig. 1 




Fig. 2 




Fig. 3 



PLATE 2 
The Influence of Heated Soils on Seed Germination and Plant Growth 

Fig. 1. Growth of soy beans and tobacco on Waukesha silt loam soil heated to different 
temperatures, showing the beneficial action of the lower and higher temperatures of heating 
on soybeans, but the retarded recovery at 150°-250°, whereas the tobacco has recovered and 
shows beneficial action at these temperatures. y4,Not heated; B, heated to 50°C.; C, 100°C.; 
D, 150°C.; E, 200°C.; F, 250°C.; G, 300°C.; H, 350°C. 

Fig. 2. Showing the variation in behavior of different crops on virgin sandy loam soil heated 
to 115°C. A, Tomatoes on unheated soil; B, tomatoes on heated soil; C, wheat on unhealed 
soil; D, wheat on heated soil; E, buckwheat on unheated soil; F, buckwheat on heated soil 



90 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 

JAMES JOHNSON 



PLATE 2 








Fig. 2 



91 



1 



PLATE 3 

The Influence of Heated Soils on Seed Germination and Plant Growth 

Fig. 1. The growth of tomatoes on muck soil heated for different lengths of time at 115°C., 
showing an appreciable beneficial action at the longer times of heating. 1, Not heated; 2 
heated 10 minutes; 3, 20 minutes; 4, 40 minutes; 5, 80 minutes; 6, 160 minutes. 

Fig. 2. The growth of tomatoes on virgin sandy loam heated for different lengths of time 
at 115°C. showing a decided injurious action at the longer times of heating. A, Not heated; 
B, heated 10 minutes; C, 20 minutes; D, 40 minutes; E, 80 minutes; F, 160 minutes. 



92 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 

JAMES JOHNSON 



PLATE 3 




Fig. 1 



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Fig. 2 



93 



PLATE 4 
The Influence of Heated Soils on Seed Germination and Plant Growth 

Fig. 1. Showing the influence of soil temperature on the extent of the toxic action of heated 
Miami silt loam soil on the growth of tobacco. lA, Not heated and grown at 28-29°C.; JB, 
heated to 110°C. and grown at28-29°C.; 2.1, not heated and grown at 23-2 4°C.; 25, heated 
to ] 10°C. and grown at 23-24°C. 

Fig. 2. Showing the influence of soil temperature on the extent of the toxic action of 
x'irgin sandy loam on the growth of tomatoes. lA, Not sterilized soil and grown at 17- 
18°C.; IB, sterilized soil and grown at 17-18°C.; 2/1, not sterilized soil and grown at 23-24°C.; 
2B, sterilized soil and grown at 23-24°C.; 3A, not sterilized soil and grown at 28-29°C.; 3B. 
sterilized soil and grown at 28-29°C. 



I 



94 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 

JAMES JOHNSON 



PLATE 4 




cr^ 



^Jfe. 



H ^* li 




Fig. 1 






Fig. 2 



95 



PLATE 5 
The Influence of Heated Soils on Seed Germination and Plant Growth 

Fig. 1. Illustrating the influence of soil temperature on the toxic action of heated soil on 
the roots of tomato plants. The higher soil temperatures greatly reduced the extent of the 
toxic action. 

Fig. 2. "Chemical" injury of heated greenhouse compost (manure and sod mixture) on 
tomato plant. This injury occurred suddenly after plant had been growing normally for 
some time. A, Check, soil not heated; B, soil heated to 110°C. 



I 



96 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 

JAMES JOHNSON 



PLATE 5 




Fig. 1 




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Fig. 2 
97 



i 



PLATE 6 

The Influence of Heated Soils on Seed Germination and Plant Growth 

"Chemical" injuries to the foliage of various plants as a result of growing on heat steri- 
lized soils. A, Mottled leaf of tomatoes quite common on certain heated soils; B, collapse of 
leaflets and petiole of tomato grown on heated soil; C, "leaf spots" of tobacco; D, marginal 
spotting on soybeans; £, injury to midribs and veins of leaflets of soybeans resulting in curl- 
ing; F, "specking" of cowpeas. 



( 



98 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 

JAMES JOHNSON 



PLATE 6 





B 




99 



1 



PLATE 7 
The Influence of Heated Soils on Seed Germination and Plant Growth 

Fig. 1. The growth of Pyroncma on soil heated to different temperatures. A, Not heated; 
B, heated to 50°C.; C, 100°C.; D, 150°C.; E, 200°C.; F, 250°C.; G, 300°C.; //, 350°C. 

Fig. 2. Growth of tomatoes on silt loam soil heated to different temperatures, as com- 
pared with treatment with varying amounts of ammonia, as ammonium carbonate, illustrating 
the similarity in responses obtainable, i^. Not heated; 2^4, heated to 150°C.; J/1, heated 
to 200°C.; 4A, heated to 250°C.; IB, no treatment; 2B, treated with 0.1 per cent ammonium 
carbonate; 3B, treated with 0.2 per cent ammonium carbonate; 4B, treated with 0.4 per 
cent ammonium carbonate. 



100 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 

JAMES JOHNSON 



PLATE 7 




Fig. 1 





Fig. 2 



101 



PLATE 8 
The Influence of Heated Soils on Seed Germination and Plant Growth 

Fig. 1. Growth of wheat on fine sandy loam under various conditions with respect to 
activity of microorganisms. A, Sterilized soil reinoculated with normal soil flora; B, 
setrilized soil under aseptic conditions; C, unsterilized soil check. 

Fig. 2. Showing the relative development of tops and roots of wheat grown on heated 
and unheated soil under varying conditions with respect to activity of microorganisms. A, 
Sterilized soil reinoculated with normal soil flora; B, sterilized soil, and aseptic conditions; 
C, unsterilized soil check. Note especially the stunted root systems in B, but better aerial 
growth than in C in spite of the toxic action. The loss of the toxic action on roots in rein- 
oculated soil is shown by comparing A with B. 



102 



INFLUENCE OF HEATED SOILS ON GERMINATION AND GROWTH 

JAMES JOHNSON 



PLATE 8 



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Fig. 1 





Fig 2 
103 






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